U.S. patent application number 10/552552 was filed with the patent office on 2006-08-17 for methods and means for increasing the tolerance of plants to stress conditions.
This patent application is currently assigned to Bayer BioScience N.V.. Invention is credited to Marc De Block.
Application Number | 20060185038 10/552552 |
Document ID | / |
Family ID | 33161008 |
Filed Date | 2006-08-17 |
United States Patent
Application |
20060185038 |
Kind Code |
A1 |
De Block; Marc |
August 17, 2006 |
Methods and means for increasing the tolerance of plants to stress
conditions
Abstract
Methods and means are provided to increase the tolerance of
plants to abiotic stress or adverse growing conditions, including
drought, high light intensities, high temperatures, nutrient
limitations and the like by reducing the activity of endogenous
PARG proteins in plants.
Inventors: |
De Block; Marc; (Merelbeke,
BE) |
Correspondence
Address: |
HUNTON & WILLIAMS LLP;INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W.
SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Assignee: |
Bayer BioScience N.V.
IP Department Technolgiepark 38
Gent
BE
B-9052
|
Family ID: |
33161008 |
Appl. No.: |
10/552552 |
Filed: |
April 9, 2004 |
PCT Filed: |
April 9, 2004 |
PCT NO: |
PCT/EP04/03995 |
371 Date: |
October 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60496588 |
Aug 21, 2003 |
|
|
|
Current U.S.
Class: |
800/289 ;
435/468 |
Current CPC
Class: |
C12N 9/0028 20130101;
C12N 15/8271 20130101; C12Y 105/01011 20130101; C12Y 302/01143
20130101 |
Class at
Publication: |
800/289 ;
435/468 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C12N 15/82 20060101 C12N015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 9, 2003 |
EP |
03076044.1 |
Claims
1. A method of producing a plant tolerant to stress conditions
comprising the steps of (a) providing plant cells with a chimeric
gene to create transgenic plant cells, said chimeric gene
comprising the following operably linked DNA fragments (i) a
plant-expressible promoter; (ii) a DNA region, which when
transcribed yields an ParG inhibitory RNA molecule; (iii) a 3' end
region involved in transcription termination and polyadenylation;
(b) regenerating a population of transgenic plant lines from said
transgenic plant cell; and (c) identifying a stress tolerant plant
line within said population of transgenic plant lines.
2. The method according to claim 1, wherein said parG inhibitory
RNA molecule comprises a nucleotide sequence of at least 20
consecutive nucleotides of the nucleotide sequence of the ParG gene
present in said plant cell.
3. The method according to claim 1, wherein said parG inhibitory
RNA molecule comprises a nucleotide sequence of at least 20
consecutive nucleotides of the complement of the nucleotide
sequence of the ParG gene present in said plant cell.
4. The method according to claim 2 or 3, wherein said chimeric gene
further comprises a DNA region encoding a self-splicing ribozyme
between said DNA region coding for said parG inhibitory RNA
molecule and said 3' end region.
5. The method according to claim 1, wherein said parG inhibitory
RNA comprises a sense region comprising a nucleotide sequence of at
least 20 consecutive nucleotides of the nucleotide sequence of the
ParG gene present in said plant cell and an antisense region
comprising a nucleotide sequence of at least 20 consecutive
nucleotides of the complement of the nucleotide sequence of the
ParG gene present in said plant cell, wherein said sense and
antisense region are capable of forming a double stranded RNA
region comprising said at least 20 consecutive nucleotides.
6. The method according to claim 1, wherein said stress conditions
are heat, drought, nutrient depletion, oxidative stress or high
light conditions.
7. The method according to claim 1, comprising further crossing
said transgenic plant line with another plant line to obtain stress
tolerant progeny plants.
8. A method of producing a plant tolerant to stress conditions
comprising the steps of: (a) isolating a DNA fragment of at least
100 bp comprising a part of the parG encoding gene of said plant;
(b) producing a chimeric gene by operably linking the following DNA
fragments; (i) a plant expressible promoter region; (ii) said
isolated DNA fragment comprising part of the parG encoding gene of
said plant in direct orientation compared to the promoter region;
(iii) said isolated DNA fragment comprising part of the parG
encoding gene of said plant in inverted orientation compared to the
promoter region; (iv) a 3' end region involved in transcription
termination and polyadenylation; (c) providing plant cells with
said chimeric gene to create transgenic plant cells (d)
regenerating a population of transgenic plant lines from said
transgenic plant cell; and (e) identifying a stress tolerant plant
line within said population of transgenic plant lines.
9. A DNA molecule comprising (i) a plant-expressible promoter; (ii)
a DNA region, which when transcribed yields a ParG inhibitory RNA
molecule; and (iii) a 3' end region involved in transcription
termination and polyadenylation.
10. The DNA molecule according to claim 9, wherein said DNA region
comprises a nucleotide sequence of at least 21 to 100 nucleotides
of a nucleotide sequence encoding a protein comprising the amino
acid sequence of SEQ ID No 1, 2 or 16 or at least 21 to 100
nucleotides of a nucleotide sequence of SEQ ID 3, 4, 15 or 23.
11. A plant cell comprising the DNA molecule of claim 9 or 10.
12. A plant consisting essentially of the plant cells of claim
11.
13. A process for producing stress tolerant plants, comprising the
step of crossing a plant of claim 12 with another plant.
14. Seeds and propagating material of a plant according to claim
12.
15. Plants obtainable or obtained by the process of claim 8.
16. A method of producing a plant tolerant to stress conditions
comprising the steps of (a) providing plant cells with a chimeric
gene to create transgenic plant cells, said chimeric gene
comprising the following operably linked DNA fragments (i) a
plant-expressible promoter; (ii) a DNA region, which when
transcribed yields an ParG inhibitory RNA molecule, said DNA region
comprising a nucleotide sequence of at least 21 to 100 nucleotides
of a nucleotide sequence encoding a protein comprising the amino
acid sequence of SEQ ID No 1, 2 or 16 or at least 21 to 100
nucleotides of a nucleotide sequence of SEQ ID 3, 4, 15 or 23;
(iii) a 3' end region involved in transcription termination and
polyadenylation; (b) regenerating a population of transgenic plant
lines from said transgenic plant cell; and (c) identifying a stress
tolerant plant line within said population of transgenic plant
lines.
17. A method of producing a plant tolerant to stress conditions
comprising the steps of (a) subjecting a plant cell line or a plant
or plant line, to mutagenesis; (b) identifying those plant cells or
plants that have a mutation in an endogenous ParG gene; (c)
subjecting the identified plant cells or plants to stress
conditions; (d) identifying plant cells or plants that tolerate
said stress conditions better than control plants.
18. A method of producing a plant tolerant to stress conditions
comprising the steps of (a) selecting a plant cell line or a plant
or plant line which is resistant to a ParG inhibitor; (b)
identifying those plant cells or plants that have a mutation in an
endogenous ParG gene; (c) subjecting the identified plant cells or
plants to stress conditions; (d) identifying plant cells or plants
that tolerate said stress conditions better than control
plants.
19. A stress tolerant plant cell or plant comprising a mutation in
an endogenous ParG gene.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of poly
(ADP-ribose) glycohydrolases in plants to increase the tolerance of
plants to adverse growing conditions, including drought, high light
intensities, high temperatures, nutrient limitations and the like.
Methods and means are provided to produce plants that are tolerant
to abiotic stress conditions.
BACKGROUND TO THE INVENTION
[0002] Frequently, abiotic stress will lead either directly or
indirectly to damage of the DNA of the cells of the plants exposed
to the adverse conditions. Genomic damage, if left unrepaired, can
lead to cell death. Tolerance to stress conditions exhibited by
plants is the result of the ability of the plant cells exposed to
the adverse conditions to reduce and/or repair the damage, and to
survive.
[0003] Plant cells, like other eukaryotic cells, have evolved an
elaborate DNA repair system. The activation of poly(ADP-ribose)
polymerase (PARP) by DNA strand breaks is often one of the first
cellular responses to DNA damage. PARP catalyzes the
post-translational modification of proteins by adding successively
molecules of ADP-ribose, obtained from the conversion of
nicotineamide dinucleotide (NAD), to form multibranched polymers
containing up to 200 ADP-ribose residues (about 40 residues in
plants). The dependence of poly(ADP-ribose) synthesis on DNA strand
breaks, and the presence of PARP in multiprotein complexes further
containing key effectors of DNA repair, replication and
transcription reactions, strongly suggests that this
posttranslational modification is involved in metabolism of nucleic
acids, and DNA repair. There are also indications that poly
(ADP-ribose) synthesis is involved in regulation of cell cycle and
cell death.
[0004] Poly (ADP-ribosylation) of proteins is transient in living
cells. The poly (ADP-ribose) polymers are rapidly turned over,
being converted to free ADP-ribose by the exoglycosidase and
endoglycosidase activity of poly (ADP-ribose) glycohydrolase (PARG;
E.C.3.2.1.143). The most proximal unit of ADP ribose on the protein
acceptor is hydrolyzed by the action of another enzyme (ADP-ribosyl
protein lyase).
[0005] In addition to this positive (DNA-repair associated) effect
of PARP on cell survival, there is also a negative effect of PARP.
The process of activating PARP upon DNA damage is associated with a
rapid lowering of NAD+ levels, since each ADP-ribose unit
transferred by PARP consumes one molecule of NAD+. NAD+ depletion
in turn results in ATP depletion, because NAD+ resynthesis requires
at least (depending on the biosynthesis pathway) three molecules of
ATP per molecule of NAD+. Furthermore, NAD+ depletion block
glyceraldehyde-3-phosphate dehydrogenase activity, which is
required to resynthesize ATP during glycolysis. Finally, NAD+ is a
key carrier of electrons needed to generate ATP via electron
transport and oxidative phosphorylation.
[0006] The physiological consequence of NAD+ and ATP depletion has
been established in the context of DNA-damage induced cell death.
It has been shown that the completion of apoptosis is absolutely
dependent on the presence of ATP and that, in the absence of this
nucleotide, the type of cellular demise switches from apoptosis to
necrosis. Since the cellular lysis associates with necrosis
generates further damage to neighboring cells it is preferable for
multicellular organisms to favor apoptotic cell death rather than
necrosis.
[0007] It is thus very important to consider the delicate balance
of positive and negative effects of the poly (ADP ribosyl)ation on
the potential of a cell to survive DNA damage.
[0008] WO 00/04173 describes methods to modulate programmed cell
death (PCD) in eukaryotic cells and organisms, particularly plant
cells and plants, by introducing of "PCD modulating chimeric genes"
influencing the expression and/or apparent activity of endogenous
poly-(ADP-ribose) polymerase (PARP) genes. Programmed cell death
may be inhibited or provoked. The invention particularly relates to
the use of nucleotide sequences encoding proteins with PARP
activity for modulating PCD, for enhancing growth rate or for
producing stress tolerant cells and organisms.
[0009] PARG encoding genes have been identified in a number of
animals such as Rattus norvegicus (Accession numbers:
NM.sub.--031339, NW.sub.--043030, AB019366,), Mus musculus
(Accession numbers: NT.sub.--039598, NM.sub.--003631, AF079557),
Homo sapiens (Accession numbers: NT.sub.--017696; NM.sub.--003631,
AF005043), Bos taurus (Accession numbers: NM.sub.--174138, U78975)
Drosophila melanogaster (Accession number: AF079556)
[0010] In plants, a poly(ADP-ribose) glycohydrolase has been
identified by map-based cloning of the wild-type gene inactivated
in a mutant affected in clock-controlled transcription of genes in
Arabidopsis and in photoperiod dependent transition from vegetative
growth to flowering (tej). The nucleotide sequence of the gene can
be obtained from nucleotide databases under the accession number
AF394690 (Panda et al., 2002 Dev. Cell. 3, 51-61).
SUMMARY OF THE INVENTION
[0011] The invention provides a method to produce a plant tolerant
to stress conditions comprising the steps of providing plant cells
with a chimeric gene to create transgenic plant cells, wherein the
chimeric gene comprises the following operably linked DNA
fragments: a plant-expressible promoter; a DNA region, which when
transcribed yields an ParG inhibitory RNA molecule; and a 3' end
region involved in transcription termination and polyadenylation. A
population of transgenic plant lines is regenerated from the
transgenic plant cell; and a stress tolerant plant line is
identified within the population of transgenic plant lines. The
ParG inhibitory RNA molecule may comprise a nucleotide sequence of
at least 20 consecutive nucleotides of the nucleotide sequence of
the ParG gene present in the plant cell (the endogenous ParG gene).
The ParG inhibitory RNA molecule may also comprise a nucleotide
sequence of at least 20 consecutive nucleotides of the complement
of the nucleotide sequence of the ParG gene present in the plant
cell (the endogenous ParG gene). In yet another embodiment, the
parG inhibitory RNA may comprise a sense region comprising a
nucleotide sequence of at least 20 consecutive nucleotides of the
nucleotide sequence of the ParG gene present in the plant cell and
an antisense region comprising a nucleotide sequence of at least 20
consecutive nucleotides of the complement of the nucleotide
sequence of the ParG gene present in the plant cell, wherein the
sense and antisense region are capable of forming a double stranded
RNA region comprising said at least 20 consecutive nucleotides. The
chimeric gene may further comprise a DNA region encoding a
self-splicing ribozyme between said DNA region coding for parG
inhibitory RNA molecule and the 3' end region. Stress conditions
may be selected from heat, drought, nutrient depletion, oxidative
stress or high light conditions.
[0012] In another embodiment of the invention, a method is provided
to produce a plant tolerant to stress conditions comprising the
steps of: isolating a DNA fragment of at least 100 bp comprising a
part of the parG encoding gene of the plant of interest; producing
a chimeric gene by operably linking a plant expressible promoter
region to the isolated DNA fragment comprising part of the parG
encoding gene of the plant in direct orientation compared to the
promoter region; and to the isolated DNA fragment comprising part
of the parG encoding gene of said plant in inverted orientation
compared to the promoter region, and a 3' end region involved in
transcription termination and polyadenylation. These chimeric genes
are then provided to plant cells to create transgenic plant cells.
A population of transgenic plant lines is regenerated from the
transgenic plant cells; and a stress tolerant plant line is
identified within the population of transgenic plant lines. The
invention also relates to stress tolerant plant cells and plants
obtained by this process.
[0013] In yet another embodiment of the invention, a method is
provided to produce a plant tolerant to stress conditions
comprising the steps of providing plant cells with a chimeric gene
to create transgenic plant cells, comprising a DNA region, which
when transcribed yields an ParG inhibitory RNA molecule, whereby
the DNA region comprises a nucleotide sequence of at least 21 to
100 nucleotides of a nucleotide sequence encoding a protein
comprising the amino acid sequence of SEQ ID No 1, 2 or 16 or at
least 21 to 100 nucleotides of a nucleotide sequence of SEQ ID 3,
4, 15 or 23 operably linked to a plant-expressible promoter and a
3' end region involved in transcription termination and
polyadenylation; regenerating a population of transgenic plant
lines from said transgenic plant cell; and identifying a stress
tolerant plant line within the population of transgenic plant
lines.
[0014] The invention also provides DNA molecules comprising a
plant-expressible promoter, operably linked to a DNA region, which
when transcribed yields an ParG inhibitory RNA molecule, and to a
3' end region involved in transcription termination and
polyadenylation. The ParG inhibitory RNA molecule may comprise a
nucleotide sequence of at least 20 consecutive nucleotides of the
nucleotide sequence of the ParG gene present in the plant cell (the
endogenous ParG gene). The ParG inhibitory RNA molecule may also
comprise a nucleotide sequence of at least 20 consecutive
nucleotides of the complement of the nucleotide sequence of the
ParG gene present in the plant cell (the endogenous ParG gene). In
yet another embodiment, the parG inhibitory RNA may comprise a
sense region comprising a nucleotide sequence of at least 20
consecutive nucleotides of the nucleotide sequence of the ParG gene
present in the plant cell and an antisense region comprising a
nucleotide sequence of at least 20 consecutive nucleotides of the
complement of the nucleotide sequence of the ParG gene present in
the plant cell, wherein the sense and antisense region are capable
of forming a double stranded RNA region comprising said at least 20
consecutive nucleotides. The chimeric gene may further comprise a
DNA region encoding a self-splicing ribozyme between said DNA
region coding for parG inhibitory RNA molecule and the 3' end
region. The chimeric gene may also comprise a nucleotide sequence
of at least 21 to 100 nucleotides of a nucleotide sequence encoding
a protein comprising the amino acid sequence of SEQ ID No 1, 2 or
16 or at least 21 to 100 nucleotides of a nucleotide sequence of
SEQ ID 3, 4, 15 or 23.
[0015] In yet another embodiment, the invention relates to plant
cell comprising the DNA molecule of the invention and plants
consisting essentially of such plant cells, as well as to processes
for producing stress tolerant plants, comprising the step of
further crossing such plants with another plant. Seeds and
propagating material of such plants comprising the chimeric genes
of the invention are also provided.
[0016] The invention also relates to a method for obtaining stress
tolerant plants comprising the steps of subjecting a plant cell
line or a plant or plant line, to mutagenesis; identifying those
plant cells or plants that have a mutation in an endogenous ParG
gene; subjecting the identified plant cells or plants to stress
conditions and identifying plant cells or plants that tolerate said
stress conditions better than control plants. Alternatively, plant
cells or plants may be selected for resistance to ParG inhibitors
and further treated as described in this paragraph.
[0017] The invention further relates to a stress tolerant plant
cell or plant having a mutation in the endogenous ParG gene.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1. Schematic representation of the poly-ADP ribose
polymeratization/depolymerization cycle by the action of PARP/PARG
in a eukaryotic cell.
[0019] FIG. 2. Diagram of the NAD+ and ATP content of Arabidopsis
lines under high light stress. Dark boxes represent NAD content
under high light conditions expressed as percentage of the value
for NAD content determined under low light conditions. Light boxes
represent ATP content under high light conditions expressed as
percentage of the value for ATP content determined under low light
conditions.
[0020] FIG. 3. Diagram of the NAD+ and ATP content of corn lines
under nitrogen depletion stress. Dark boxes represent NAD content
while light boxes represent ATP content.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0021] The invention is based, on the one hand, on the
demonstration that cells from stress resistant plant lines
comprising a chimeric gene reducing the PARP gene expression,
exhibited a higher NAD/ATP content under adverse conditions than
cells from untransformed plant lines. On the other hand, it has
been observed that silencing of the expression of PARG encoding
gene in tobacco using a transient silencing RNA vector based on
satellite viruses resulted in a similar phenotype as that observed
for silencing of PARP encoding gene using the same silencing
system. Furthermore, silencing the expression of PARG encoding gene
in plants, such as Arabidopsis and tobacco, resulted in plants that
were more resistant to stress conditions, such as e.g. those
imposed by high light conditions.
[0022] Although not intending to limit the invention to a specific
mode of action, it is expected that silencing of PARG gene
expression results in a similar phenotype as silencing of PARP gene
expression for the following reasons. As can be seen from FIG. 1,
polymerization of ADP ribose catalyzed by PARP, consuming NAD, is
followed by depolymerization of poly ADP ribose, catalyzed by PARG.
Poly ADP ribosylation of the PARP protein itself results in
inactivation of the PARP protein. The speed at which the ADP ribose
polymerization/depolymerization cycle occurs in plant cells,
leading to NAD depletion and consequently ATP depletion, can be
slowed down or stopped by reduction of the PARP gene expression or
of the enzymatic activity of PARP. As a result, plant cells, and
plants comprising such cells are more resistant to adverse
conditions. The data provided here indicate that a similar effect
can be obtained through slowing down or stopping the cycle by
reduction of the PARG gene expression or PARG activity.
[0023] The invention relates to reduction of plant cell death in
response to adverse environmental conditions, and consequently to
enhanced stress resistance, by altering the level of expression of
ParG genes, or by altering the activity or the apparent activity of
PARG proteins in that plant cell. Conveniently, the level of
expression of ParG genes may be controlled genetically by
introduction of chimeric genes altering the expression of ParG
genes, or by altering the endogenous PARG encoding genes, including
the expression signals.
[0024] In one embodiment of the invention, a method for producing
plants tolerant to stress conditions or adverse growing conditions
is provided comprising the steps of:
[0025] providing plant cells with a chimeric gene to create
transgenic plant cells, wherein the chimeric gene comprises the
following operably linked DNA fragments: [0026] a plant-expressible
promoter; [0027] a DNA region, which when transcribed yields a ParG
inhibitory RNA molecule; [0028] a 3' end region involved in
transcription termination and polyadenylation;
[0029] regenerating a population of transgenic plant lines from
said transgenic plant cell; and
[0030] identifying a stress tolerant plant line within said
population of transgenic plant lines.
[0031] As used herein "a stress tolerant plant" or "a plant
tolerant to stress conditions or adverse growing conditions" is a
plant (particularly a plant obtained according to the methods of
the invention), which, when subjected to adverse growing conditions
for a period of time, such as but not limited to drought, high
temperatures, limited supply of nutrients (particularly nitrogen),
high light intensities, grows better than a control plant not
treated according to the methods of the invention. This will
usually be apparent from the general appearance of the plants and
may be measured e.g., by increased biomass production, continued
vegetative growth under adverse conditions or higher seed yield.
Stress tolerant plant have a broader growth spectrum, i.e. they are
able to withstand a broader range of climatological and other
abiotic changes, without yield penalty. Biochemically, stress
tolerance may be apparent as the higher NAD.sup.+-NADH/ATP content
and lower production of reactive oxygen species of stress tolerant
plants compared to control plants under stress condition. Stress
tolerance may also be apparent as the higher chlorophyll content,
higher photosynthesis and lower chlorophyll fluorescence under
stress conditions in stress tolerant plants compared to control
plants under the same conditions.
[0032] It will be clear that it is also not required that the plant
be grown continuously under the adverse conditions for the stress
tolerance to become apparent. Usually, the difference in stress
tolerance between a plant or plant cell according to the invention
and a control plant or plant cell will become apparent even when
only a relatively short period of adverse conditions is encountered
during growth.
[0033] As used herein, a "ParG inhibitory RNA molecule" is an RNA
molecule that is capable of decreasing the expression of the
endogenous PARG encoding genes of a plant cell, preferably through
post-transcriptional silencing. It will be clear that even when a
ParG inhibitory RNA molecule decreases the expression of a PARG
encoding gene through post-transcriptional silencing, such an RNA
molecule may also exert other functions within a cell, such as e.g.
guiding DNA methylation of the endogenous ParG gene, again
ultimately leading to decreased expression of the PARG encoding
gene. Also, expression of the endogenous PARG encoding genes of a
plant cell may be reduced by transcriptional silencing, e.g., by
using RNAi or dsRNA targeted against the promoter region of the
endogenous ParG gene.
[0034] As used herein, a "PARG encoding gene" or a "ParG gene" is a
gene capable of encoding a PARG (poly ADP ribose glycohydrolase)
protein, wherein the PARG protein catalyzes the depolymerization of
poly ADP-ribose, by releasing free ADP ribose units either by
endoglycolytic or exoglycolytic action.
[0035] PARG encoding genes may comprise a nucleotide sequence
encoding a protein comprising the amino acid sequence of SEQ ID No
1 (Arabidopsis thaliana) or of SEQ ID No 2 (Solanum tuberosum) or
of SEQ ID No 16 (Oryza sativa) or parts thereof, such as a DNA
fragment comprising the nucleotide sequence of SEQ ID No 3 or SEQ
ID 4 or SEQ ID No 15. or SEQ ID 23 (Zea mays).
[0036] However, it will be clear that the skilled person can
isolate variant DNA sequences from other plant species, by
hybridization with a probe derived from the above mentioned PARG
encoding genes from plant species, or even with a probe derived
from the above mentioned PARG encoding genes from animal species.
To this end, the probes should preferably have a nucleotide
sequence comprising at least 40 consecutive nucleotides from the
coding region of those mentioned PARG encoding genes sequences,
preferably from the coding region of SEQ ID No 3 or SEQ ID No 4.
The probes may however comprise longer regions of nucleotide
sequences derived from the ParG genes, such as about 50, 60, 75,
100, 200 or 500 consecutive nucleotides from any of the mentioned
ParG genes. Preferably, the probe should comprise a nucleotide
sequence coding for one of the highly conserved regions of the
catalytic domain, which have been identified by aligning the
different PARG proteins from animals. These regions are also
present in the identified PARG protein from Arabidopsis thaliana
and comprise the amino acid sequence LXVDFANXXXGGG (corresponding
to SEQ ID No 1 from the amino acid at position 252 to the amino
acid at position 264; X may be any amino acid)
LXVDFANXXXGGGXXXXGXVQEEIRF (corresponding to SEQ ID No 1 from the
amino acid at position 252 to the amino acid at position 277) or
LXVDFANXXXGGGXXXXGXVQEEIRFXXXPE (corresponding to SEQ ID No 1 from
the amino acid at position 252 to the amino acid at position 282),
TGXWGCGXFXGD (corresponding to SEQ ID No 1 from the amino acid at
position 449 to the amino acid at position 460) or
TGXWGCGAFXGDXXLKXXXQ (corresponding to SEQ ID No 1 from the amino
acid at position 449 to the amino acid at position 468). Other
conserved regions have the amino acid sequence DXXXRXXXXAIDA
(corresponding to SEQ ID No 1 from the amino acid at position 335
to the amino acid at position 344) or REXXKAXXGF (corresponding to
SEQ ID No 1 from the amino acid at position 360 to the amino acid
at position 369) or GXXXXSXYTGY (corresponding to SEQ ID No 1 from
the amino acid at position 303 to the amino acid at position 313).
Hybridization should preferably be under stringent conditions.
[0037] "Stringent hybridization conditions" as used herein mean
that hybridization will generally occur if there is at least 95%
and preferably at least 97% sequence identity between the probe and
the target sequence. Examples of stringent hybridization conditions
are overnight incubation in a solution comprising 50% formamide,
5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared carrier DNA such as
salmon sperm DNA, followed by washing the hybridization support in
0.1.times.SSC at approximately 65.degree. C., e.g. for about 10 min
(twice). Other hybridization and wash conditions are well known and
are exemplified in Sambrook et al, Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor, N.Y. (1989),
particularly chapter 11.
[0038] Alternatively, ParG encoding genes or parts thereof may also
be isolated by PCR based techniques, using as primers
oligonucleotides comprising at least 20 consecutive nucleotides
from a nucleotide sequence of the mentioned PARG encoding genes or
the complement thereof. Such primers may comprise a nucleotide
sequence encoding a conserved region, as mentioned above, or be
complementary to such a nucleotide sequence. Oligonucleotides which
may be used for that purpose may comprise the nucleotide sequence
of either or SEQ ID No. 5, SEQ ID No 6., SEQ ID No. 7 or SEQ ID No.
8. Oligonucleotides which may be used may also be degenerate, such
as the oligonucleotide primers of SEQ ID No 17, SEQ ID No 18, SEQ
ID No 19; SEQ ID No 20, SEQ ID No 21 or SEQ ID No 22.
[0039] Specific PCR fragments from ParG genes may e.g., be obtained
by using combinations of the oligonucleotides having the nucleotide
sequence of SEQ ID No. 5 and SEQ ID No 6 using e.g., Arabidopsis
genomic DNA or cDNA as a template DNA, or by using combinations of
the oligonucleotides having the nucleotide sequence of SEQ ID No. 7
and SEQ ID No 8 using e.g., potato genomic DNA or cDNA as a
template DNA, under stringent annealing conditions.
[0040] The isolated sequences may encode a functional PARG protein
or a part thereof. Preferably the isolated sequences should
comprise a nucleotide sequence coding for one or more of the highly
conserved regions from the catalytic domain of PARG proteins as
mentioned elsewhere.
[0041] However, for the purpose of the invention is not required
that the isolated sequences encode a functional ParG protein nor
that a complete coding region is isolated. Indeed, all that is
required for the invention is that a chimeric gene can be designed
or produced, based on the identified or isolated sequence of the
endogenous ParG gene from a plant, which is capable of producing a
ParG inhibitory RNA. Several alternative methods are available to
produce such a ParG inhibitory RNA molecule.
[0042] In one embodiment, the ParG inhibitory RNA molecule encoding
chimeric gene is based on the so-called antisense technology. In
other words, the coding region of the chimeric gene comprises a
nucleotide sequence of at least 20 consecutive nucleotides of the
complement of the nucleotide sequence of the endogenous ParG gene
of the plant cell or plant, the expression of which is targeted to
be reduced. Such a chimeric gene may be conveniently constructed by
operably linking a DNA fragment comprising at least 20 nucleotides
from the isolated or identified ParG gene, or part of such a gene,
in inverse orientation, to a plant expressible promoter and 3'end
formation region involved in transcription termination and
polyadenylation. It will be immediately clear that there is no need
to know the exact nucleotide sequence or complete nucleotide
sequence of such a DNA fragment from an isolated ParG gene.
[0043] In another embodiment the ParG inhibitory RNA molecule
encoding chimeric gene is based on the so-called co-suppression
technology. In other words, the coding region of the chimeric gene
comprises a nucleotide sequence of at least 20 consecutive
nucleotides of the nucleotide sequence of the endogenous ParG gene
of the plant cell or plant, the expression of which is targeted to
be reduced. Such a chimeric gene may be conveniently constructed by
operably linking a DNA fragment comprising at least 20 nucleotides
from the isolated or identified ParG gene, or part of such a gene,
in direct orientation, to a plant expressible promoter and 3'end
formation region involved in transcription termination and
polyadenylation. Again it is not required to know the exact
nucleotide sequence of the used DNA fragment from the isolated ParG
gene.
[0044] The efficiency of the above mentioned chimeric genes in
reducing the expression of the endogenous ParG gene may be further
enhanced by inclusion of DNA elements which result in the
expression of aberrant, unpolyadenylated ParG inhibitory RNA
molecules. One such DNA element suitable for that purpose is a DNA
region encoding a self-splicing ribozyme, as described in WO
00/01133.
[0045] The efficiency or the above mentioned chimeric genes in
reducing the expression of the endogenous ParG gene of a plant cell
may also be further enhanced by including into one plant cell
simultaneously a chimeric gene as herein described encoding a
antisense ParG inhibitory RNA molecule and a chimeric gene as
herein described encoding a sense ParG inhibitory RNA molecule,
wherein said antisense and sense ParG inhibitory RNA molecules are
capable of forming a double stranded RNA region by base pairing
between the mentioned at least 20 consecutive nucleotides, as
described in WO 99/53050.
[0046] As further described in WO 99/53050, the sense and antisense
ParG inhibitory RNA regions, capable of forming a double stranded
RNA region may be present in one RNA molecule, preferably separated
by a spacer region. The spacer region may comprise an intron
sequence. Such a chimeric gene may be conveniently constructed by
operably linking a DNA fragment comprising at least 20 nucleotides
from the isolated or identified endogenous ParG gene, the
expression of which is targeted to be reduced, in an inverted
repeat, to a plant expressible promoter and 3' end formation region
involved in transcription termination and polyadenylation. To
achieve the construction of such a chimeric gene, use can be made
of the vectors described in WO 02/059294
[0047] An embodiment of the invention thus concerns a method for
obtaining a stress tolerant plant line comprising the steps of
[0048] providing plant cells with a chimeric gene to create
transgenic plant cells, wherein the chimeric gene comprises the
following operably linked DNA fragments: [0049] a plant-expressible
promoter, [0050] a DNA region, which when transcribed yields a ParG
inhibitory RNA molecule comprising a nucleotide sequence of at
least 20 consecutive nucleotides of the nucleotide sequence of the
ParG gene present in said plant cell; or [0051] a DNA region, which
when transcribed yields a ParG inhibitory RNA molecule comprising a
nucleotide sequence of at least 20 consecutive nucleotides of the
complement of the nucleotide sequence of the ParG gene present in
said plant cell; or [0052] a DNA region, which when transcribed
yields a ParG inhibitory RNA molecule comprising a sense region
comprising a nucleotide sequence of at least 20 consecutive
nucleotides of the nucleotide sequence of the ParG gene present in
said plant cell and an antisense region comprising a nucleotide
sequence of at least 20 consecutive nucleotides of the complement
of the nucleotide sequence of the ParG gene present in said plant
cell, wherein said sense and antisense region are capable of
forming a double stranded RNA region comprising said at least 20
consecutive nucleotides. [0053] a 3' end region involved in
transcription termination and polyadenylation;
[0054] regenerating a population of transgenic plant lines from
said transgenic plant cell; and
[0055] identifying a stress tolerant plant line within said
population of transgenic plant lines.
[0056] As used herein "comprising" is to be interpreted as
specifying the presence of the stated features, integers, steps or
components as referred to, but does not preclude the presence or
addition of one or more features, integers, steps or components, or
groups thereof. Thus, e.g., a nucleic acid or protein comprising a
sequence of nucleotides or amino acids, may comprise more
nucleotides or amino acids than the actually cited ones, i.e., be
embedded in a larger nucleic acid or protein. A chimeric gene
comprising a DNA region which is functionally or structurally
defined, may comprise additional DNA regions etc.
[0057] It will thus be clear that the minimum nucleotide sequence
of the antisense or sense RNA region of about 20 nt of the ParG
coding region may be comprised within a larger RNA molecule,
varying in size from 20 nt to a length equal to the size of the
target gene.
[0058] The mentioned antisense or sense nucleotide regions may thus
be about from about 21 nt to about 5000 nt long, such as 21 nt, 40
nt, 50 nt, 100 nt, 200 nt, 300 nt, 500 nt, 1000 nt, 2000 nt or even
about 5000 nt or larger in length.
[0059] Moreover, it is not required for the purpose of the
invention that the nucleotide sequence of the used inhibitory ParG
RNA molecule or the encoding region of the chimeric gene, is
completely identical or complementary to the endogenous ParG gene
the expression of which is targeted to be reduced in the plant
cell. The longer the sequence, the less stringent the requirement
for the overall sequence identity is. Thus, the sense or antisense
regions may have an overall sequence identity of about 40% or 50%
or 60% or 70% or 80% or 90% or 100% to the nucleotide sequence of
the endogenous ParG gene or the complement thereof. However, as
mentioned antisense or sense regions should comprise a nucleotide
sequence of 20 consecutive nucleotides having about 100% sequence
identity to the nucleotide sequence of the endogenous ParG gene.
Preferably the stretch of about 100% sequence identity should be
about 50, 75 or 100 nt.
[0060] For the purpose of this invention, the "sequence identity"
of two related nucleotide sequences, expressed as a percentage,
refers to the number of positions in the two optimally aligned
sequences which have identical residues (.times.100) divided by the
number of positions compared. A gap, i.e. a position in an
alignment where a residue is present in one sequence but not in the
other is regarded as a position with non-identical residues. The
alignment of the two sequences is performed by the Needleman and
Wunsch algorithm (Needleman and Wunsch 1970) Computer-assisted
sequence alignment, can be conveniently performed using standard
software program such as GAP which is part of the Wisconsin Package
Version 10.1 (Genetics Computer Group, Madison, Wis., USA) using
the default scoring matrix with a gap creation penalty of 50 and a
gap extension penalty of 3.
[0061] It will be clear that whenever nucleotide sequences of RNA
molecules are defined by reference to nucleotide sequence of
corresponding DNA molecules, the thymine (T) in the nucleotide
sequence should be replaced by uracil (U). Whether reference is
made to RNA or DNA molecules will be clear from the context of the
application.
[0062] It will also be clear that chimeric genes capable of
producing inhibitory ParG genes for a particular ParG gene in a
particular plant variety or plant species, may also be used to
inhibit ParG gene expression in other plant varieties or plant
species. Indeed, when sufficient homology exists between the ParG
inhibitory RNA region and the ParG gene, or when the ParG genes
share the same stretch of 19 nucleotides, expression of those other
genes will also be down-regulated.
[0063] In view of the potential role of ParG in nucleic acid
metabolism, it may be advantageous that the expression of the
endogenous ParG gene by the ParG inhibitory RNA is not completely
inhibited. Downregulating the expression of a particular gene by
gene silencing through the introduction of a chimeric gene encoding
ParG inhibitory RNA will result in a population of different
transgenic lines, exhibiting a distribution of different degrees of
silencing of the ParG gene. The population will thus contain
individual transgenic plant lines, wherein the endogenous ParG gene
is silenced to the required degree of silencing. A person skilled
in the art can easily identify such plant lines, e.g. by subjecting
the plant lines to a particular adverse condition, such a high
light intensity, oxidative stress, drought, heat etc. and selecting
those plants which perform satisfactory and survive best the
treatment.
[0064] As used herein, the term "promoter" denotes any DNA which is
recognized and bound (directly or indirectly) by a DNA-dependent
RNA-polymerase during initiation of transcription. A promoter
includes the transcription initiation site, and binding sites for
transcription initiation factors and RNA polymerase, and can
comprise various other sites (e.g., enhancers), at which gene
expression regulatory proteins may bind.
[0065] The term "regulatory region", as used herein, means any DNA,
that is involved in driving transcription and controlling (i.e.,
regulating) the timing and level of transcription of a given DNA
sequence, such as a DNA coding for a protein or polypeptide. For
example, a 5' regulatory region (or "promoter region") is a DNA
sequence located upstream (i.e., 5') of a coding sequence and which
comprises the promoter and the 5'-untranslated leader sequence. A
3' regulatory region is a DNA sequence located downstream (i.e.,
3') of the coding sequence and which comprises suitable
transcription termination (and/or regulation) signals, including
one or more polyadenylation signals.
[0066] In one embodiment of the invention the promoter is a
constitutive promoter. In another embodiment of the invention, the
promoter activity is enhanced by external or internal stimuli
(inducible promoter), such as but not limited to hormones, chemical
compounds, mechanical impulses, abiotic or biotic stress
conditions. The activity of the promoter may also regulated in a
temporal or spatial manner (tissue-specific promoters;
developmentally regulated promoters).
[0067] For the purpose of the invention, the promoter is a
plant-expressible promoter. As used herein, the term
"plant-expressible promoter" means a DNA sequence which is capable
of controlling (initiating) transcription in a plant cell. This
includes any promoter of plant origin, but also any promoter of
non-plant origin which is capable of directing transcription in a
plant cell, i.e., certain promoters of viral or bacterial origin
such as the CaMV35S (Hapster et al., 1988), the subterranean clover
virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters
but also tissue-specific or organ-specific promoters including but
not limited to seed-specific promoters (e.g., WO89/03887),
organ-primordia specific promoters (An et al., 1996), stem-specific
promoters (Keller et al., 1988), leaf specific promoters (Hudspeth
et al., 1989), mesophyl-specific promoters (such as the
light-inducible Rubisco promoters), root-specific promoters (Keller
et al., 1989), tuber-specific promoters (Keil et al., 1989),
vascular tissue specific promoters (Peleman et al., 1989),
stamen-selective promoters (WO 89/10396, WO 92/13956), dehiscence
zone specific promoters (WO 97/13865) and the like.
[0068] Methods for the introduction of chimeric genes into plants
are well known in the art and include Agrobacterium-mediated
transformation, particle gun delivery, microinjection,
electroporation of intact cells, polyethyleneglycol-mediated
protoplast transformation, electroporation of protoplasts,
liposome-mediated transformation, silicon-whiskers mediated
transformation etc. The transformed cells obtained in this way may
then be regenerated into mature fertile plants.
[0069] The transgenic plant cells and plant lines according to the
invention may further comprise chimeric genes which will reduce the
expression of PARP genes as described in WO 00/04173. These further
chimeric genes may be introduced e.g. by crossing the transgenic
plant lines of the current invention with transgenic plants
containing PARP gene expression reducing chimeric genes. Transgenic
plant cells or plant lines may also be obtained by introducing or
transforming the chimeric genes of the invention into transgenic
plant cells comprising the PARP gene expression reducing chimeric
genes or vice versa. Alternatively, the PARP and PARG inhibitory
RNA regions may be encoded by one chimeric gene and transcribed as
one RNA molecule.
[0070] The chimeric genes of the invention (or the inhibitory RNA
molecules corresponding thereto) may also be introduced into plant
cells in a transient manner, e.g. using the viral vectors, such as
viral RNA vectors as described in WO 00/63397 or WO 02/13964.
[0071] Having read this specification, it will be immediately clear
to the skilled artisan, that mutant plant cells and plant lines,
wherein the PARG activity is reduced may be used to the same effect
as the transgenic plant cells and plant lines described herein.
Mutants in ParG gene of a plant cell or plant may be easily
identified using screening methods known in the art, whereby
chemical mutagenesis, such as e.g., EMS mutagenesis, is combined
with sensitive detection methods (such as e.g., denaturing HPLC).
An example of such a technique is the so-called "Targeted Induced
Local Lesions in Genomes" method as described in McCallum et al,
Plant Physiology 123 439-442 or WO 01/75167. However, other methods
to detect mutations in particular genome regions or even alleles,
are also available and include screening of libraries of existing
or newly generated insertion mutant plant lines, whereby pools of
genomic DNA of these mutant plant lines are subjected to PCR
amplification using primers specific for the inserted DNA fragment
and primers specific for the genomic region or allele, wherein the
insertion is expected (see e.g. Maes et al., 1999, Trends in Plant
Science, 4, pp 90-96).
[0072] Plant cell lines and plant lines may also be subjected to
mutagenesis by selection for resistance to ParG inhibitors, such as
gallotannines. (Ying, et al. (2001). Proc. Natl. Acad. Sci. USA
98(21), 12227-12232; Ying, W., Swanson, R. A. (2000). NeuroReport
11 (7), 1385-1388.
[0073] Thus, methods are available in the art to identify plant
cells and plant lines comprising a mutation in the ParG gene. This
population of mutant cells or plant lines can then be subjected to
different abiotic stresses, and their phenotype or survival can be
easily determined. Additionally, the NAD and/or the ATP content of
the stressed cells can be determined and compared to results of
such determinations of unstressed cells. In stress tolerant cells,
the reduction of NAD content under stress conditions should when
compared with unstressed cells, should be lower than for
corresponding control cells.
[0074] It is also an object of the invention to provide plant cells
and plants containing the chimeric genes or the RNA molecules
according to the invention. Gametes, seeds, embryos, either zygotic
or somatic, progeny or hybrids of plants comprising the chimeric
genes of the present invention, which are produced by traditional
breeding methods are also included within the scope of the present
invention.
[0075] The plants obtained by the methods described herein may be
further crossed by traditional breeding techniques with other
plants to obtain stress tolerant progeny plants comprising the
chimeric genes of the present invention.
[0076] The methods and means described herein are believed to be
suitable for all plant cells and plants, both dicotyledonous and
monocotyledonous plant cells and plants including but not limited
to cotton, Brassica vegetables, oilseed rape, wheat, corn or maize,
barley, alfalfa, peanuts, sunflowers, rice, oats, sugarcane,
soybean, turf grasses, barley, rye, sorghum, sugar cane, vegetables
(including chicory, lettuce, tomato, zucchini, bell pepper,
eggplant, cucumber, melon, onion, leek), tobacco, potato,
sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, but also
plants used in horticulture, floriculture or forestry (poplar, fir,
eucalyptus etc.).
[0077] The following non-limiting Examples describe method and
means for increasing stress tolerance in plants according to the
invention.
[0078] Unless stated otherwise in the Examples, all recombinant DNA
techniques are carried out according to standard protocols as
described in Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and
in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in
Molecular Biology, Current Protocols, USA. Standard materials and
methods for plant molecular work are described in Plant Molecular
Biology Labfax (1993) by R. D. D. Croy, jointly published by BIOS
Scientific Publications Ltd (UK) and Blackwell Scientific
Publications, UK. Other references for standard molecular biology
techniques include Sambrook and Russell (2001) Molecular Cloning: A
Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory
Press, NY, Volumes I and II of Brown (1998) Molecular Biology
LabFax, Second Edition, Academic Press (UK). Standard materials and
methods for polymerase chain reactions can be found in Dieffenbach
and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, and in McPherson at al. (2000)
PCR--Basics: From Background to Bench, First Edition, Springer
Verlag, Germany.
[0079] Throughout the description and Examples, reference is made
to the following sequences:
[0080] SEQ ID No. 1: amino acid sequence of the ParG protein from
Arabidopsis thaliana.
[0081] SEQ ID No. 2: amino acid sequence of part of the ParG
protein from Solanum tuberosum.
[0082] SEQ ID No. 3: nucleotide sequence encoding the ParG protein
from Arabidopsis thaliana.
[0083] SEQ ID No. 4: nucleotide sequence encoding the part of the
ParG protein from Solanum tuberosum.
[0084] SEQ ID No. 5: nucleotide sequence of an oligonucleotide
primer suitable for PCR amplification of part of a ParG protein
encoding DNA fragment.
[0085] SEQ ID No. 6: nucleotide sequence of an oligonucleotide
primer suitable for PCR amplification of part of a ParG protein
encoding DNA fragment.
[0086] SEQ ID No. 7: nucleotide sequence of an oligonucleotide
primer suitable for PCR amplification of part of a ParG protein
encoding DNA fragment.
[0087] SEQ ID No. 8: nucleotide sequence of an oligonucleotide
primer suitable for PCR amplification of part of a ParG protein
encoding DNA fragment.
[0088] SEQ ID No. 9: nucleotide sequence of the T-DNA vector
containing the ParG expression reducing chimeric gene based on the
Arabidopsis ParG gene sequence.
[0089] SEQ ID No. 10: amino acid sequence of conserved sequence 1
of PARG proteins.
[0090] SEQ ID No. 11: amino acid sequence of conserved sequence 2
of PARG proteins.
[0091] SEQ ID No. 12: amino acid sequence of conserved sequence 3
of PARG proteins.
[0092] SEQ ID No. 13: amino acid sequence of conserved sequence 4
of PARG proteins.
[0093] SEQ ID No. 14: amino acid sequence of conserved sequence 5
of PARG proteins.
[0094] SEQ ID No. 15: nucleotide sequence of the ParG protein from
Oryza sativa.
[0095] SEQ ID No. 16: amino acid sequence of the ParG protein from
Oryza sativa.
[0096] SEQ ID No. 17: nucleotide sequence of an oligonucleotide
primer PG1 suitable for PCR amplification of part of a ParG protein
encoding DNA fragment.
[0097] SEQ ID No. 18: nucleotide sequence of an oligonucleotide
primer PG2 suitable for PCR amplification of part of a ParG protein
encoding DNA fragment.
[0098] SEQ ID No. 19: nucleotide sequence of an oligonucleotide
primer PG3 suitable for PCR amplification of part of a ParG protein
encoding DNA fragment.
[0099] SEQ ID No. 20: nucleotide sequence of an oligonucleotide
primer PG4 suitable for PCR amplification of part of a ParG protein
encoding DNA fragment.
[0100] SEQ ID No. 21: nucleotide sequence of an oligonucleotide
primer PG5 suitable for PCR amplification of part of a ParG protein
encoding DNA fragment.
[0101] SEQ ID No. 22: nucleotide sequence of an oligonucleotide
primer PG6 suitable for PCR amplification of part of a ParG protein
encoding DNA fragment.
[0102] SEQ ID No. 23: nucleotide sequence encoding a ParG protein
from Zea mays.
[0103] SEQ ID No. 24: nucleotide sequence of a T-DNA vector
comprising a chimeric gene capable of reducing PARG expression
[0104] SEQ ID No. 25: nucleotide sequence of a T-DNA vector
comprising a chimeric gene capable of reducing PARG expression
EXAMPLES
Example 1
Analysis of the Influence of Stress on Energy Production Efficiency
of Transgenic Stress Tolerant Plant Lines Containing PARP Gene
Expresssion Reducing Chimeric Genes
[0105] Hypocotyls of transgenic Brassica napus plants comprising
PARP gene expression reducing chimeric genes as described in WO
00/04173 were cultivated for 5 days on a growth medium. Explants
were then transferred to liquid medium comprising 30 mg/L aspirin
or acetylsalicylic acid (resulting in oxidative stress conditions)
for one day. In control experiments, hypocotyls of non-transgenic
Brassica napus plants N90-740 were cultivated on the same growth
medium and then incubated for one day in liquid medium comprising
30 mg/L aspirin. In addition, hypocotyls of both the transgenic
lines and the control line were cultivated on the same growth
medium without aspirin.
[0106] After the cultivation period, the ATP content of 125
explants was determined for each experiment. Additionally, the
oxygen consumed in 3 hours by 125 explants was determined. The
results are summarized in Table 1. The standard error of the mean
was less than 6%. Whereas, the ratio of moles ATP per mg consumed
oxygen in the control plants decreased in the control plants when
oxidative stress was applied, the same ratio in the stress tolerant
transgenic plant lines actually increased under stress conditions,
and was considerably higher (about 24%) than in the control plants.
The stress resistant transgenic lines thus maintained an constant
energy production efficiency, whereas the control lines exhibited
an decreased energy production efficiency. In addition, superoxide
production, expressed as a percentage of superoxide production in
control plants not subjected to the oxidative stress, did not
increase in stress tolerant plants subjected to stress conditions.
TABLE-US-00001 TABLE 1 Influence of stress on energy production
efficiency of 5 days cultured Brassica napus hypocotyl explants.
O.sub.2 moles mg/L ATP moles consumed mg ATP in 3 hrs con- per 125
by 125 sumed Superoxide Plant line Stress explants explants O.sub.2
production N90-740 None 12.4 .times. 2.96 4.19 .times. 100%
(control) 10.sup.-7 10.sup.-7 30 mg/L 13.2 .times. 4.06 3.25
.times. 167% aspirin 10.sup.-7 10.sup.-7 Transgenic None 9.3
.times. 2.33 3.99 .times. 108% line 10.sup.-7 10.sup.-7 30 mg/L
11.4 .times. 2.82 4.04 .times. 100% aspirin 10.sup.-7 10.sup.-7
[0107] In another experiment, the NAD+ and ATP content of 4
different transgenic Arabidopsis lines comprising PARP gene
expression reducing chimeric genes as described in WO 00/04173 were
determined under high and low light conditions, and compared to the
values obtained for a non transformed control line under the same
conditions. The 4 different lines exhibited different degrees of
stress resistance as exhibited e.g. by their ability to withstand
heat and/or drought conditions. The values obtained for the NAD and
ATP contents under high light stress are expressed as a percentage
of the values for the NAD and ATP contents under low light
conditions, and are plotted in FIG. 2.
[0108] The results show that high light stress leads to a
significant NAD reduction in control plant cells and in the
transgenic plant line which is the least stress resistant. The more
stress resistant the transgenic plant lines are, the less
signicifant the NAD reduction is under high light stress
conditions.
[0109] In another experiment, the NAD+ and ATP content of a
segregating population resulting from a cross between transgenic
corn lines comprising PARP gene expression reducing chimeric genes
as described in WO 00/04173 and an untransformed corn line, were
determined under conditions of nutrient (nitrogen) depletion, and
compared to the values obtained for a non transformed control line
under the same conditions. FIG. 3 is a graphic representation of
the of the obtained results. Hemizygous and azygous lines were
discriminated by verification for the presence of the selectable
marker gene. The NAD and ATP content was significantly higher in
the hemizygous, stress tolerant plants than in the untransformed
control plants or the azygous plants.
Example 2
Construction of ParG Gene Expression Reducing Chimeric Genes
[0110] To reduce the expression of the PARG gene e.g. in
Arabidopsis and related plants, a chimeric gene was constructed
which is capable expressing a dsRNA comprising both a sense and
antisense region which can form a double stranded RNA. Such dsRNA
is very effective in reducing the expression of the genes with
which is shares sequence homology, by post-transcriptional
silencing. The chimeric gene comprises the following DNA fragments:
[0111] A promoter region from Cauliflower mosaic Virus (CaMV 35S);
[0112] A DNA fragment comprising 163 bp from the ParG gene from
Arabidopsis thaliana in direct orientation (Genbank Accession
number AF394690 from nucleotide position 973 to 1135); [0113] A DNA
fragment encoding intron 2 from the pdk gene from Flaveria; [0114]
The DNA fragment comprising 163 bp from the ParG gene from
Arabidopsis thaliana in inverted orientation (Genbank Accession
number AF394690 from nucleotide position 973 to 1135) [0115] A
fragment of the 3' untranslated end from the octopine synthetase
gene from Agrobacterium tumefaciens.
[0116] This chimeric gene was introduced in a T-DNA vector, between
the left and right border sequences from the T-DNA, together with a
selectable marker gene providing resistance to the herbicide
phosphinotricin.
[0117] To reduce the expression of the PARG gene e.g. in potatoes
and related plants, a chimeric gene is constructed which is capable
expressing a dsRNA comprising both a sense and antisense region of
a cDNA sequence from potato, that is capable of encoding a protein
having high sequence identity with the N-terminal part of the
Arabidopsis PARG protein. The chimeric gene comprises the following
DNA fragments: [0118] A promoter region from Cauliflower mosaic
Virus (CaMV 35S); [0119] A DNA fragment comprising a sequence of at
least 100 bp from ParG homologue from Solanum tuberosum in direct
orientation (Genbank Accession number BE340510); [0120] A DNA
fragment encoding intron 2 from the pdk gene from Flaveria; [0121]
The DNA fragment comprising the sequence of at least 100 bp from
ParG homologue from Solanum tuberosum in inverted orientation
(Genbank Accession number BE340510); [0122] A fragment of the 3'
untranslated end from the octopine synthetase gene from
Agrobacterium tumefaciens
[0123] This chimeric gene is introduced in a T-DNA vector, between
the left and right border sequences from the T-DNA, together with a
selectable marker gene providing resistance to the herbicide
phosphinotricin.
Example 3
Analysis of Transgenic Plant Lines Comprising ParG Gene Expression
Reducing Chimeric Genes
[0124] The chimeric genes of Example 2 are introduced into
Arabidopsis or potato respectively, by Agrobacterium mediated
transformation.
[0125] The population of obtained transgenic lines is subjected to
the following stress conditions, together with control plants:
[0126] Increased heat for a period of days (greenhouse) or hours
(in vitro) [0127] Drought for a period of days [0128] High light
conditions for a period of days [0129] Nutrient depletion
[0130] Individual plant lines surviving well the above mendioned
stress conditions are selected.
[0131] The NAD content and ATP content for the above mentioned
plants is determined under control and stress conditions.
Example 4
Quantitative Determination of NAD, ATP and Superoxide Radicals in
Plant Cells
[0132] Quantification of ATP in plant tissues was done basically as
decribed by Rawyler et al. (1999), Plant Physiol. 120, 293-300. The
assay was used for the determination of the ATP content of
hypocotyl explants that were cultured for 4-5 days on A2S3 medium
or 2 weeks old in vitro cultured Arabidopsis plants. All
manipulations are performed on crushed ice unless otherwhise
indicated.
[0133] ATP Extraction [0134] Freeze plant material with liquid
nitrogen [0135] 100 hypocotyl explants [0136] .+-.700 mg
Arabidopsis plants (roots+shoots) (about 32-37 18-days old C24
plants) [0137] Put frozen hypocotyls in mortar and add 6 ml of 6%
perchloric acid. [0138] Extraction can be done at room temperature
using a pestle. After extraction, put samples as soon as possible
on ice. [0139] Centrifuge at 24,000 g (Sorvall, SS34 rotor at
14,000 rpm) for 10 min. at 4.degree. C. [0140] The supernatant is
neutralized with 5M K.sub.2CO.sub.3 (add 350 .mu.l of 5M
K.sub.2CO.sub.3 to 3 ml of supernatant). [0141] KClO.sub.4 is
removed by spinning as described above.
[0142] Quantitative bioluminescent determination of ATP [0143] The
ATP bioluminescent assay kit from Sigma is used (FL-M). [0144]
Dilute extract 6000.times.(about 6 mL extract from which 100 .mu.l
is taken, that is diluted 1000 times) The dilutions are made with
the `ATP assay mix dilution buffer` (FL-AAB) of the ATP
bioluminescent assay kit [0145] The amount of light that is
produced is measured with the TD-20/20 luminometer of Turner
Designs (Sunnyvale, USA). [0146] Standard curve: disolve ATP
standard of kit (FL-MS) in 10 ml of water (2.times.10.sup.-6
moles)
[0147] Quantification of NAD+ and NADH in plant tissues was
performed, essentially as described by Karp et al. (1983) or
Filipovic et al. (1999) on the following plant material: [0148]
Brassica napus: 150 5-days cultured hypocotyl explants/sample
Arabidopsis: 1000 mg 18-days old in vitro grown plants
(shoots+roots)/sample (corresponds to .+-.60 C24 plants)
[0149] Assay Solution [0150] (A) For measuring NADH: [0151] 25 mM
potassium phosphate buffer pH7 [0152] 0.1 mM DTT [0153] 3 .mu.M FMN
(Fluka, 83810) [0154] 30 .mu.M n-decanal (Sigma, D-7384) [0155] (B)
For measuring NAD.sup.++NADH: [0156] idem as for measuring NADH
alone+2 .mu.g/mL alcohol dehydrogenase (Roche, 102 717)
[0157] Extraction [0158] Freeze with liquid nitrogen [0159] Put
frozen plant material in cooled mortar (cooled at -20.degree. C.)
and add 5 mL extraction buffer [0160] Grind material using a pestle
[0161] Centrifuge at 24 000 g (Sorvall, SS34 rotor at 14 000 rpm)
for 15 minutes at 4.degree. C. [0162] Take 1 mL of supernatant for
analysis
[0163] Assay
NADH
[0164] 390 .mu.L of assay solution A [0165] +10 .mu.L extract
[0166] +2 .mu.L NAD(P)H:FMN oxidoreductase [0167] +100 .mu.L
luciferase solution NAD.sup.++NADH [0168] 390 .mu.L of assay
solution B [0169] +10 .mu.L extract [0170] 2 minutes at room
temperature [0171] +2 .mu.L NAD(P)H:FMN oxidoreductase [0172] +100
.mu.L luciferase solution The amount of light that is produced is
measured with the TD-20/20 luminometer of Turner Designs
(Sunnyvale, USA) NADH-Standard
[0173] NADH stock solution: 1 mM (7.1 mg/10 mL H.sub.2O) [0174]
NADH: disodium salt, Roche, 107 735 [0175] Dilution series in 10 mM
potassium phosphate buffer pH7: (10.sup.-2); 5.times.10.sup.-3;
[0176] 2.times.10.sup.-3; 10.sup.-3; 5.times.10.sup.-4 [0177] Add
10 .mu.L of dilutions in 390 .mu.L of assay solution A and perform
reaction [0178] Make standard curve
[0179] Superoxide radicals production was measured by quantifying
the reduction of XTT as described in De Block and De Brouwer (2002)
Plant Physiol. Biochem. 40, 845-852
[0180] Brassica Napus
[0181] Media and Reaction Buffers
Sowing Medium (Medium 201):
[0182] Half concentrated Murashige and Skoog salts [0183] 2%
sucrose [0184] pH 5.8 [0185] 0.6% agar (Difco Bacto Agar) [0186]
250 mg/l triacillin Callus Inducing Medium A2S3: [0187] MS medium,
0.5 g/l Mes (pH 5.8), 3% sucrose, 40 mg/ adenine-SO.sub.4, [0188]
0.5% agarose, 1 mg/l 2,4-D, 0.25 mg/l NAA, 1 mg/l BAP, 250 mg/l
triacillin Incubation Medium: [0189] 25 mM K-phosphate buffer pH5.8
[0190] 2% sucrose [0191] 1 drop Tween20 for 25 ml medium Reaction
Buffer [0192] 50 mM K-phosphate buffer pH7.4 [0193] 1 mM sodium,
3'-{1-[phenylamino-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)=XTT
(bts, Germany, cat no. 2525) 1 drop Tween20 for 25 ml buffer
[0194] Sterilization of seeds--pregermination of seeds--growing of
the seedlings. Seeds are soaked in 70% ethanol for 2 min, then
surface-sterilized for 15 min in a sodium hypochlorite solution
(with about 6% active chlorine) containing 0.1% Tween20. Finally,
the seeds are rinsed with 1l of sterile tap water. Incubate seeds
for at least one hour in sterile tap water (to allow diffusion from
seeds of components that may inhibit germination). Seeds are put in
250 ml erlenmeyer flasks containing 50 ml of sterile tap water
(+250 mg/l triacillin). Shake for about 20 hours. Seeds from which
the radicle is protruded are put in Vitro Vent containers from
Duchefa containing about 125 ml of sowing medium (10 seeds/vessel,
not too many to reduce loss of seed by contamination). The seeds
are germinated at .+-.94.degree. C. and 10-30:Einstein/s.sup.-1
m.sup.-2 with a daylength of 16 h.
[0195] Preculture of the hypocotyl explants and induction of stress
[0196] 12-14 days after sowing, the hypocotyls are cut in about
7-10 mm segments. [0197] The hypocotyl explants (25
hypocotyls/Optilux Petridish, Falcon S1005, Denmark) are cultured
for 5 days on medium A2S3 at 25.degree. C. (at 10-30.quadrature.
Einstein/s.sup.-1m.sup.-2).
[0198] XTT-Assay [0199] Transfer 150 hypocotyl explants to a 50 ml
Falcon tube. [0200] Wash with reaction buffer (without XTT). [0201]
Add 20 mL reaction buffer+XTT. [0202] (explants have to be
submerged, but do not vacuum infiltrate) [0203] Incubate in the
dark at 26.degree. C. for about 3 hours [0204] Measure the
absorption of the reaction medium at 470 nm
[0205] Arabidopsis Thaliana
[0206] Media and reaction buffers
Plant Medium:
[0207] Half concentrated Murashige and Skoog salts [0208] B5
vitamins [0209] 1.5% sucrose [0210] pH 5.8 [0211] 0.7% Difco agar
Incubation Medium: [0212] 10 mM K-phosphate buffer pH5.8 [0213] 2%
sucrose [0214] 1 drop Tween20 for 25 ml medium Reaction Buffer:
[0215] 50 mM K-phosphate buffer pH7.4 [0216] 1 mM sodium,
3'-{1-[phenylamino-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)=XTT
(bts, Germany, cat no. 2525) [0217] 1 drop Tween20 for 25 ml
buffer
[0218] Arabidopsis Plants [0219] Arabidopsis lines: [0220] control
[0221] lines to test [0222] Sterilization of Arabidopsis seeds:
[0223] 2 min. 70% ethanol [0224] 10 min. bleach (6% active
chlorine)+1 drop Tween 20 for 20 ml [0225] solution [0226] wash 5
times with sterile tap water [0227] Pregermination of seeds: [0228]
In 9 cm Optilux Petridishes (Falcon) containing 12 ml sterile tap
water. [0229] Low light overnight to 24 hours. [0230] Growing of
Arabidopsis plants [0231] Seeds are sown in Intergrid Tissue
Culture disks of Falcon (nr. 3025) containing.+-.125 ml of plant
medium: 1 seed/grid. [0232] Plants are grown at 24.degree. C.
[0233] 30.mu.Einstein s.sup.-1m.sup.-2 [0234] 16 hours light-8
hours dark [0235] for about 3 weeks (before bolting)
[0236] XTT-Assay
Control Condition (No Stress)
[0237] Harvest shoots (roots included) from agar plates and put
them directly in a 50 ml Falcon tube containing reaction buffer
(without XTT) Stressed Shoots [0238] Transfer shoots to 50 ml
Falcon tubes containing reaction buffer (without XTT) [0239]
Replace reaction buffer with buffer containing XTT (40 mL/tube)
[0240] Shoots have to be submerged, but do not vacuum infiltrate
[0241] Incubate in the dark at 26.degree. C. for about 3 hours
[0242] Measure the absorption of the reaction medium at 470 nm
[0243] Quantification of respiration by measuring oxygen
consumption using a Clark polarographic electrode was done in the
following way:
[0244] Plant Material
Brassica napus
[0245] 150-200* hypocotyl explants Cultured for 5 days at
25.degree. C. [0246] (cfr. protocol vigour assay) [0247] * 150
explants error<10%; 200 explants error<6%
[0248] Arabidopsis [0249] For C24.+-.1000 mg* in vitro plants
(shoots+roots) (corresponds with [0250] .about.50 18-days old
plants) [0251] Pregerminate seeds before sowing [0252] Grow for 18
days at 24.degree. C. [0253] (cfr. protocol in vitro growth
Arabidopis) [0254] * for error<8%
[0255] Incubation Media
Brassica napus
[0256] 25 mM K-phosphate buffer pH5.8 [0257] 2% sucrose [0258]
Tween20 (1 drop/25 ml) Arabidopsis [0259] 10 mM K-phosphate buffer
pH5.8 [0260] 2% sucrose [0261] Tween20 (1 drop/25 ml) Before use,
aerate (saturate with oxygen) medium well by stirring for at least
a few hours
[0262] Assay [0263] Put explants in 100 ml glass bottle (Schott,
Germany) filled with incubation medium. Put the same weight of
shoots in each bottle (+700 mg) [0264] Fill bottle to overflowing
and close tightly (avoid large air bubbles) [0265] Fill also a
bottle with incubation medium that does not contain explants
(blanco) [0266] Incubate at 24.degree. C. at low light for: [0267]
34 hours (Brassica napus) [0268] 3 hours (Arabidopsis) [0269] Shake
gently during incubation (to avoid oxygen depletion of medium
around explants) [0270] Measure oxygen concentration (mg/l) of
incubation media using an hand-held dissolved oxygen meter
(Cyberscan DO 310; Eutech Instruments, Singapore) [0271] mg/l
consumed oxygen=[oxygen]blanco-[oxygen]sample.
Example 5
Analysis of Transgenic Plant Lines Comprising ParG Gene Expression
Reducing Chimeric Genes
[0272] The chimeric genes of Example 2 were introduced into
Arabidopsis an Nicotiana tabacum c.v. Petit Havana SR1 by
Agrobacterium mediated transformation.
[0273] Transgenic seeds were germinated on a medium containing MS
salts/2; B5 vitamins; 1.5% sucrose; pH5.8 and 0.7% Difco agar.
Germinated seeds were subject to low light (photosynthetic photon
flux of about 30 .mu.mol m.sup.-1 s.sup.-1 for 14 to 18 days, after
which the light intensity was increased about 6-fold
(photosynthetic photon flux of about 190 .mu.mol m.sup.-1
s.sup.-1). After 1 day, the NAD and NADH contents were determined
using the enzymatic cycling method (Karp et al. (1983) Anal.
Biochem. 128, pp 175-180). A portion of the seedlings were
cultivated further under high light conditions for about 3 to about
days, after which the damage was scored. Damage was visible as
darkening of the young leaves and shoot tip, bleaching of older
leaves and growth retardation. The results are summarized in Table
1 for Arabidopsis and in Table 2 for tobacco. TABLE-US-00002 TABLE
1 Analysis of Arabidopsis (Columbia). % NAD + NADH TTC-reducing
High light content in 1 gram capacity vs tolerance of tissue
(10.sup.-3 .mu.M) control Non-transgenic control S 17.3 100
Transgenic line 9 R 28.2 ND Transgenic line 10 R 31.7 ND Transgenic
line 11 .+-.R 26.5 ND Transgenic line 12 S 19.4 ND Transgenic line
26 R 33.2 55 Transgenic line 27 S 21.3 100 Transgenic line 28 .+-.R
26.5 75 Transgenic line 29 S 17.7 102 Transgenic line 30 R 28.3 66
.+-.R indicates that some dark pigmentation was observed. ND: not
determined
[0274] TABLE-US-00003 TABLE 2 Analysis of Nicotiana tabacum c.v.
Petit Havana SR1. % TTC-reducing High light capacity vs tolerance
control Non-transgenic control S 100 Transgenic line 1 R/S 88
Transgenic line 2 .+-.R 79 Transgenic line 3 R 53 .+-.R indicates
that some dark pigmentation was observed. R/S indicates tha the
resistance phenotype was not very clear.
[0275] There is a positive correlation between the resistance to
high light stress in the transgenic plants and the NAD+NADH content
of the cells. An inverse correlation can be seen between TTC
reducing capacity and high light tolerance.
Example 6
Construction of ParG Gene Expression Reducing Chimeric Genes Suited
for Use in Cereal Plants
[0276] To reduce the expression of the PARG gene e.g. in cereals
such as rice or corn (maize) and related plants, a chimeric gene is
constructed which is capable expressing a dsRNA comprising both a
sense and antisense region of nucleotide sequence from rice, that
is capable of encoding a protein having high sequence identity with
PARG protein encoding nucleotide sequences. The chimeric gene
comprises the following DNA fragments: [0277] A promoter region
from Cauliflower mosaic Virus (CaMV 35S); [0278] A DNA fragment
comprising a sequence of at least 100 bp from ParG homologue from
Oryza saliva (SEQ ID No 15) in direct orientation; [0279] A DNA
fragment encoding intron 2 from the pdk gene from Flaveria; [0280]
A DNA fragment comprising a sequence of at least 100 bp from ParG
homologue from Oryza sativa (SEQ ID No 15) in inverted orientation;
[0281] A fragment of the 3' untranslated end from the octopine
synthetase gene from Agrobacterium tumefaciens.
[0282] This chimeric gene is introduced in a T-DNA vector, between
the left and right border sequences from the T-DNA, together with a
selectable marker gene providing resistance to e.g. the herbicide
phosphinotricin.
[0283] To reduce the expression of the PARG gene e.g. in cereals
such as rice or corn (maize) and related plants, a chimeric gene is
constructed which is capable expressing a dsRNA comprising both a
sense and antisense region of nucleotide sequence from rice, that
is capable of encoding a protein having high sequence identity with
PARG protein encoding nucleotide sequences. The chimeric gene
comprises the following DNA fragments: [0284] A promoter region
from Cauliflower mosaic Virus (CaMV 35S); [0285] A DNA fragment
comprising a sequence of at least 100 bp from ParG homologue from
Zea mays (SEQ ID No 23) in direct orientation; [0286] A DNA
fragment encoding intron 2 from the pdk gene from Flaveria; [0287]
A DNA fragment comprising a sequence of at least 100 bp from ParG
homologue from Zea mays (SEQ ID No 23) in inverted orientation;
[0288] A fragment of the 3' untranslated end from the octopine
synthetase gene from Agrobacterium tumefaciens.
[0289] This chimeric gene is introduced in a T-DNA vector, between
the left and right border sequences from the T-DNA, together with a
selectable marker gene providing resistance to e.g. the herbicide
phosphinotricin. The nucleotide sequence of two examples of such
T-DNA vectors comprising two different chimeric gences as described
in the previous paragraph is represented in SEQ ID Nos 24 and
25.
Example 7
Analysis of Transgenic Plant Lines Comprising ParG Gene Expression
Reducing Chimeric Genes
[0290] The chimeric genes of Example 6 are introduced into rice or
corn respectively, by Agrobacterium mediated transformation.
[0291] The population of obtained transgenic lines is subjected to
the following stress conditions, together with control plants:
[0292] Increased heat for a period of days (greenhouse) or hours
(in vitro) [0293] Drought for a period of days [0294] High light
conditions for a period of days [0295] Nutrient depletion
[0296] Individual plant lines surviving well the above mentioned
stress conditions, or at least one thereof, are selected.
[0297] The NAD content and ATP content for the above mentioned
plants is determined under control and stress conditions.
Sequence CWU 1
1
25 1 548 PRT Arabidopsis thaliana 1 Met Glu Asn Arg Glu Asp Leu Asn
Ser Ile Leu Pro Tyr Leu Pro Leu 1 5 10 15 Val Ile Arg Ser Ser Ser
Leu Tyr Trp Pro Pro Arg Val Val Glu Ala 20 25 30 Leu Lys Ala Met
Ser Glu Gly Pro Ser His Ser Gln Val Asp Ser Gly 35 40 45 Glu Val
Leu Arg Gln Ala Ile Phe Asp Met Arg Arg Ser Leu Ser Phe 50 55 60
Ser Thr Leu Glu Pro Ser Ala Ser Asn Gly Tyr Ala Phe Leu Phe Asp 65
70 75 80 Glu Leu Ile Asp Glu Lys Glu Ser Lys Arg Trp Phe Asp Glu
Ile Ile 85 90 95 Pro Ala Leu Ala Ser Leu Leu Leu Gln Phe Pro Ser
Leu Leu Glu Val 100 105 110 His Phe Gln Asn Ala Asp Asn Ile Val Ser
Gly Ile Lys Thr Gly Leu 115 120 125 Arg Leu Leu Asn Ser Gln Gln Ala
Gly Ile Val Phe Leu Ser Gln Glu 130 135 140 Leu Ile Gly Ala Leu Leu
Ala Cys Ser Phe Phe Cys Leu Phe Pro Asp 145 150 155 160 Asp Asn Arg
Gly Ala Lys His Leu Pro Val Ile Asn Phe Asp His Leu 165 170 175 Phe
Ala Ser Leu Tyr Ile Ser Tyr Ser Gln Ser Gln Glu Ser Lys Ile 180 185
190 Arg Cys Ile Met His Tyr Phe Glu Arg Phe Cys Ser Cys Val Pro Ile
195 200 205 Gly Ile Val Ser Phe Glu Arg Lys Ile Thr Ala Ala Pro Asp
Ala Asp 210 215 220 Phe Trp Ser Lys Ser Asp Val Ser Leu Cys Ala Phe
Lys Val His Ser 225 230 235 240 Phe Gly Leu Ile Glu Asp Gln Pro Asp
Asn Ala Leu Glu Val Asp Phe 245 250 255 Ala Asn Lys Tyr Leu Gly Gly
Gly Ser Leu Ser Arg Gly Cys Val Gln 260 265 270 Glu Glu Ile Arg Phe
Met Ile Asn Pro Glu Leu Ile Ala Gly Met Leu 275 280 285 Phe Leu Pro
Arg Met Asp Asp Asn Glu Ala Ile Glu Ile Val Gly Ala 290 295 300 Glu
Arg Phe Ser Cys Tyr Thr Gly Tyr Ala Ser Ser Phe Arg Phe Ala 305 310
315 320 Gly Glu Tyr Ile Asp Lys Lys Ala Met Asp Pro Phe Lys Arg Arg
Arg 325 330 335 Thr Arg Ile Val Ala Ile Asp Ala Leu Cys Thr Pro Lys
Met Arg His 340 345 350 Phe Lys Asp Ile Cys Leu Leu Arg Glu Ile Asn
Lys Ala Leu Cys Gly 355 360 365 Phe Leu Asn Cys Ser Lys Ala Trp Glu
His Gln Asn Ile Phe Met Asp 370 375 380 Glu Gly Asp Asn Glu Ile Gln
Leu Val Arg Asn Gly Arg Asp Ser Gly 385 390 395 400 Leu Leu Arg Thr
Glu Thr Thr Ala Ser His Arg Thr Pro Leu Asn Asp 405 410 415 Val Glu
Met Asn Arg Glu Lys Pro Ala Asn Asn Leu Ile Arg Asp Phe 420 425 430
Tyr Val Glu Gly Val Asp Asn Glu Asp His Glu Asp Asp Gly Val Ala 435
440 445 Thr Gly Asn Trp Gly Cys Gly Val Phe Gly Gly Asp Pro Glu Leu
Lys 450 455 460 Ala Thr Ile Gln Trp Leu Ala Ala Ser Gln Thr Arg Arg
Pro Phe Ile 465 470 475 480 Ser Tyr Tyr Thr Phe Gly Val Glu Ala Leu
Arg Asn Leu Asp Gln Val 485 490 495 Thr Lys Trp Ile Leu Ser His Lys
Trp Thr Val Gly Asp Leu Trp Asn 500 505 510 Met Met Leu Glu Tyr Ser
Ala Gln Arg Leu Tyr Lys Gln Thr Ser Val 515 520 525 Gly Phe Phe Ser
Trp Leu Leu Pro Ser Leu Ala Thr Thr Asn Lys Ala 530 535 540 Ile Gln
Pro Pro 545 2 169 PRT Solanum tuberosum 2 Met Glu Asn Arg Glu Asp
Val Lys Ser Ile Leu Pro Phe Leu Pro Val 1 5 10 15 Cys Leu Arg Ser
Ser Ser Leu Phe Trp Pro Pro Leu Val Val Glu Ala 20 25 30 Leu Lys
Ala Leu Ser Glu Gly Pro His Tyr Ser Asn Val Asn Ser Gly 35 40 45
Gln Val Leu Phe Leu Ala Ile Ser Asp Ile Arg Asn Ser Leu Ser Leu 50
55 60 Pro Asp Ser Ser Ile Ser Ser Ser Ala Ser Asp Gly Phe Ser Leu
Leu 65 70 75 80 Phe Asp Asp Leu Ile Pro Arg Asp Glu Ala Val Lys Trp
Phe Lys Glu 85 90 95 Val Val Pro Lys Met Ala Asp Leu Leu Leu Arg
Leu Pro Ser Leu Leu 100 105 110 Glu Ala His Tyr Glu Lys Ala Asp Gly
Gly Ile Val Lys Gly Val Asn 115 120 125 Thr Gly Leu Arg Leu Leu Glu
Ser Gln Gln Pro Gly Ile Val Phe Leu 130 135 140 Ser Gln Glu Leu Val
Gly Ala Leu Leu Ala Cys Ser Phe Phe Cys Tyr 145 150 155 160 Ser Leu
Pro Met Ile Glu Val Ser Val 165 3 1647 DNA Arabidopsis thaliana 3
atggagaatc gcgaagatct taactcaatt cttccgtacc ttccacttgt aattcgttcg
60 tcgtcgctgt attggccgcc gcgtgtggtg gaggcgttaa aggcaatgtc
tgaaggacca 120 tctcacagcc aagttgactc aggagaggtt ctacggcaag
ctattttcga tatgagacga 180 tccttatctt tctctactct cgagccatct
gcttctaatg gctacgcatt tctctttgac 240 gaattgattg atgagaaaga
atcaaagaga tggttcgatg agattatccc agcattggcg 300 agcttacttc
tacagtttcc atctctgtta gaagtgcatt tccaaaatgc tgataatatt 360
gttagtggaa tcaaaaccgg tcttcgtttg ttaaattccc aacaagctgg cattgttttc
420 ctcagccagg agttgattgg agctcttctt gcatgctctt tcttttgttt
gtttccggat 480 gataatagag gtgcaaaaca ccttccagtc atcaactttg
atcatttgtt tgcaagcctt 540 tatataagtt atagtcaaag tcaagaaagc
aagataagat gtattatgca ttactttgaa 600 aggttttgct cctgcgtgcc
tattggtatt gtttcatttg aacgcaagat taccgctgct 660 cctgatgctg
atttctggag caagtctgac gtttctcttt gtgcatttaa ggttcactct 720
tttgggttaa ttgaagatca acctgacaat gctctcgaag tggactttgc aaacaagtat
780 ctcggaggtg gttccctaag tagagggtgc gtgcaggaag agatacgctt
catgattaac 840 cctgaattaa tcgctggcat gcttttcttg cctcggatgg
atgacaatga agctatagaa 900 atagttggtg cggaaagatt ttcatgttac
acagggtatg catcttcgtt tcggtttgct 960 ggtgagtaca ttgacaaaaa
ggcaatggat cctttcaaaa ggcgaagaac cagaattgtt 1020 gcaattgatg
cattatgtac accgaagatg agacacttta aagatatatg tcttttaagg 1080
gaaattaata aggcactatg tggcttttta aattgtagca aggcttggga gcaccagaat
1140 atattcatgg atgaaggaga taatgaaatt cagcttgtcc gaaacggcag
agattctggt 1200 cttctgcgta cagaaactac tgcgtcacac cgaactccac
taaatgatgt tgagatgaat 1260 agagaaaagc ctgctaacaa tcttatcaga
gatttttatg tggaaggagt tgataacgag 1320 gatcatgaag atgatggtgt
cgcgacaggg aattggggat gtggtgtttt tggaggagac 1380 ccagagctaa
aggctacgat acaatggctt gctgcttccc agactcgaag accatttata 1440
tcatattaca cctttggagt agaggcactc cgaaacctag atcaggtgac gaagtggatt
1500 ctttcccata aatggactgt tggagatctg tggaacatga tgttagaata
ttctgctcaa 1560 aggctctaca agcaaaccag tgttggcttc ttttcttggc
tacttccatc tctagctacc 1620 accaacaaag ctatccagcc gccttga 1647 4 598
DNA Solanum tuberosum 4 gcaatggaga atagagaaga cgtgaagtca atccttccct
ttttgccggt gtgtctccga 60 tcatcttctc ttttctggcc gccgctagtt
gttgaagcac tgaaagccct ctctgaaggc 120 cctcattaca gcaatgttaa
ctccggccaa gtcctcttcc tcgcaatctc cgacattcgg 180 aattcccttt
cactacctga ttcttcaatt tcctcttctg cttcagacgg attttctctc 240
ttatttgatg atttaattcc tagggatgaa gctgttaaat ggttcaaaga agtggtgccg
300 aaaatggcgg atttgctatt gcggttgcct tccttattgg aggctcacta
tgagaaggct 360 gatggtggaa ttgttaaagg agtcaacact ggtcttcgct
tattggaatc acaacagcct 420 ggcattgttt tcctcagtca ggaattagtc
ggtgctcttc ttgcatgttc cttcttttgc 480 tattccctac caatgataga
ggtatctgta tgatcagtat gacgagaaat ttgaaaataa 540 attgaagtgc
attcttcact attttgagag gattggctca ttgatacctg cgggctac 598 5 37 DNA
Artificial Sequence oligonucleotide primer ParGAt1 5 ggatcccctg
caggacaaaa aggcaatgga tcctttc 37 6 39 DNA Artificial Sequence
oligonucleotide primer ParGAt2 6 gcacgaattc gcggccgcgg tgctcccaag
ccttgctac 39 7 39 DNA Artificial Sequence oligonucleotide primer
ParGSt1 7 ggatcccctg caggctcact atgagaaggc tgatggtgg 39 8 43 DNA
Artificial Sequence oligonucleotide primer ParGSt2 8 gcacgaattc
gcggccgcgt catactgatc atacagatac ctc 43 9 13466 DNA Artificial
Sequence nucleotide sequence of pTVE428 9 agattcgaag ctcggtcccg
tgggtgttct gtcgtctcgt tgtacaacga aatccattcc 60 cattccgcgc
tcaagatggc ttcccctcgg cagttcatca gggctaaatc aatctagccg 120
acttgtccgg tgaaatgggc tgcactccaa cagaaacaat caaacaaaca tacacagcga
180 cttattcaca cgcgacaaat tacaacggta tatatcctgc cagtactcgg
ccgtcgaccg 240 cggtaccccg gaattaagct tgcatgcctg caggtcctgc
tgagcctcga catgttgtcg 300 caaaattcgc cctggacccg cccaacgatt
tgtcgtcact gtcaaggttt gacctgcact 360 tcatttgggg cccacataca
ccaaaaaaat gctgcataat tctcggggca gcaagtcggt 420 tacccggccg
ccgtgctgga ccgggttgaa tggtgcccgt aactttcggt agagcggacg 480
gccaatactc aacttcaagg aatctcaccc atgcgcgccg gcggggaacc ggagttccct
540 tcagtgaacg ttattagttc gccgctcggt gtgtcgtaga tactagcccc
tggggccttt 600 tgaaatttga ataagattta tgtaatcagt cttttaggtt
tgaccggttc tgccgctttt 660 tttaaaattg gatttgtaat aataaaacgc
aattgtttgt tattgtggcg ctctatcata 720 gatgtcgcta taaacctatt
cagcacaata tattgttttc attttaatat tgtacatata 780 agtagtaggg
tacaatcagt aaattgaacg gagaatatta ttcataaaaa tacgatagta 840
acgggtgata tattcattag aatgaaccga aaccggcggt aaggatctga gctacacatg
900 ctcaggtttt ttacaacgtg cacaacagaa ttgaaagcaa atatcatgcg
atcataggcg 960 tctcgcatat ctcattaaag caggactcta gagacaaaaa
ggcaatggat cctttcaaaa 1020 ggcgaagaac cagaattgtt gcaattgatg
cattatgtac accgaagatg agacacttta 1080 aagatatatg tcttttaagg
gaaattaata aggcactatg tggcttttta aattgtagca 1140 aggcttggga
gcaccatcga tttcgaaccc agcttcccaa ctgtaatcaa tccaaatgta 1200
agatcaatga taacacaatg acatgatcta tcatgttacc ttgtttattc atgttcgact
1260 aattcattta attaatagtc aatccattta gaagttaata aaactacaag
tattatttag 1320 aaattaataa gaatgttgat tgaaaataat actatataaa
atgatagatc ttgcgctttg 1380 ttatattagc attagattat gttttgttac
attagattac tgtttctatt agtttgatat 1440 tatttgttac tttagcttgt
tatttaatat tttgtttatt gataaattac aagcagattg 1500 gaatttctaa
caaaatattt attaactttt aaactaaaat atttagtaat ggtatagata 1560
tttaattata taataaacta ttaatcataa aaaaatatta ttttaattta tttattctta
1620 tttttactat agtattttat cattgatatt taattcatca aaccagctag
aattactatt 1680 atgattaaaa caaatattaa tgctagtata tcatcttaca
tgttcgatca aattcattaa 1740 aaataatata cttactctca acttttatct
tcttcgtctt acacatcact tgtcatattt 1800 ttttacatta ctatgttgtt
tatgtaaaca atatatttat aaattatttt ttcacaatta 1860 taacaactat
attattataa tcatactaat taacatcact taactatttt atactaaaag 1920
gaaaaaagaa aataattatt tccttaccaa gctggggtac cggtgctccc aagccttgct
1980 acaatttaaa aagccacata gtgccttatt aatttccctt aaaagacata
tatctttaaa 2040 gtgtctcatc ttcggtgtac ataatgcatc aattgcaaca
attctggttc ttcgcctttt 2100 gaaaggatcc attgcctttt tgtcctcgag
cgtgtcctct ccaaatgaaa tgaacttcct 2160 tatatagagg aagggtcttg
cgaaggatag tgggattgtg cgtcatccct tacgtcagtg 2220 gagatgtcac
atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg 2280
atgctcctcg tgggtggggg tccatctttg ggaccactgt cggcagagag atcttgaatg
2340 atagcctttc ctttatcgca atgatggcat ttgtaggagc caccttcctt
ttctactgtc 2400 ctttcgatga agtgacagat agctgggcaa tggaatccga
ggaggtttcc cgaaattatc 2460 ctttgttgaa aagtctcaat agccctttgg
tcttctgaga ctgtatcttt gacatttttg 2520 gagtagacca gagtgtcgtg
ctccaccatg ttgacgaaga ttttcttctt gtcattgagt 2580 cgtaaaagac
tctgtatgaa ctgttcgcca gtcttcacgg cgagttctgt tagatcctcg 2640
atttgaatct tagactccat gcatggcctt agattcagta ggaactacct ttttagagac
2700 tccaatctct attacttgcc ttggtttatg aagcaagcct tgaatcgtcc
atactggaat 2760 agtacttctg atcttgagaa atatgtcttt ctctgtgttc
ttgatgcaat tagtcctgaa 2820 tcttttgact gcatctttaa ccttcttggg
aaggtatttg atctcctgga gattgttact 2880 cgggtagatc gtcttgatga
gacctgctgc gtaggcctct ctaaccatct gtgggtcagc 2940 attctttctg
aaattgaaga ggctaacctt ctcattatca gtggtgaaca tagtgtcgtc 3000
accttcacct tcgaacttcc ttcctagatc gtaaagatag aggaaatcgt ccattgtaat
3060 ctccggggca aaggagatct cttttggggc tggatcactg ctgggccttt
tggttcctag 3120 cgtgagccag tgggcttttt gctttggtgg gcttgttagg
gccttagcaa agctcttggg 3180 cttgagttga gcttctcctt tggggatgaa
gttcaacctg tctgtttgct gacttgttgt 3240 gtacgcgtca gctgctgctc
ttgcctctgt aatagtggca aatttcttgt gtgcaactcc 3300 gggaacgccg
tttgttgccg cctttgtaca accccagtca tcgtatatac cggcatgtgg 3360
accgttatac acaacgtagt agttgatatg agggtgttga atacccgatt ctgctctgag
3420 aggagcaact gtgctgttaa gctcagattt ttgtgggatt ggaattaatt
cgtcgagcgg 3480 ccgctcgacg agcgcgccga tatcgcgatc gcccgggccg
gccatttaaa tgaattcgag 3540 ctcggtaccc aaacgcggcc gcaagctata
acttcgtata gcatacatta tacgaagtta 3600 ttcgactcta gaggatccca
attcccatgc atggagtcaa agattcaaat agaggacact 3660 tctcgaactc
ggccgtcgaa ctcggccgtc gagtacatgg tcgataagaa aaggcaattt 3720
gtagatgtta attcccatct tgaaagaaat atagtttaaa tatttattga taaaataaca
3780 agtcaggtat tatagtccaa gcaaaaacat aaatttattg atgcaagttt
aaattcagaa 3840 atatttcaat aactgattat atcagctggt acattgccgt
agatgaaaga ctgagtgcga 3900 tattatgtgt aatacataaa ttgatgatat
agctagctta gctcatcggg ggatcctaga 3960 cgcgtgagat cagatctcgg
tgacgggcag gaccggacgg ggcggtaccg gcaggctgaa 4020 gtccagctgc
cagaaaccca cgtcatgcca gttcccgtgc ttgaagccgg ccgcccgcag 4080
catgccgcgg ggggcatatc cgagcgcctc gtgcatgcgc acgctcgggt cgttgggcag
4140 cccgatgaca gcgaccacgc tcttgaagcc ctgtgcctcc agggacttca
gcaggtgggt 4200 gtagagcgtg gagcccagtc ccgtccgctg gtggcggggg
gagacgtaca cggtcgactc 4260 ggccgtccag tcgtaggcgt tgcgtgcctt
ccaggggccc gcgtaggcga tgccggcgac 4320 ctcgccgtcc acctcggcga
cgagccaggg atagcgctcc cgcagacgga cgaggtcgtc 4380 cgtccactcc
tgcggttcct gcggctcggt acggaagttg accgtgcttg tctcgatgta 4440
gtggttgacg atggtgcaga ccgccggcat gtccgcctcg gtggcacggc ggatgtcggc
4500 cgggcgtcgt tctgggtcca ttgttcttct ttactctttg tgtgactgag
gtttggtcta 4560 gtgctttggt catctatata taatgataac aacaatgaga
acaagctttg gagtgatcgg 4620 agggtctagg atacatgaga ttcaagtgga
ctaggatcta caccgttgga ttttgagtgt 4680 ggatatgtgt gaggttaatt
ttacttggta acggccacaa aggcctaagg agaggtgttg 4740 agacccttat
cggcttgaac cgctggaata atgccacgtg gaagataatt ccatgaatct 4800
tatcgttatc tatgagtgaa attgtgtgat ggtggagtgg tgcttgctca ttttacttgc
4860 ctggtggact tggccctttc cttatgggga atttatattt tacttactat
agagctttca 4920 tacctttttt ttaccttgga tttagttaat atataatggt
atgattcatg aataaaaatg 4980 ggaaattttt gaatttgtac tgctaaatgc
ataagattag gtgaaactgt ggaatatata 5040 tttttttcat ttaaaagcaa
aatttgcctt ttactagaat tataaatata gaaaaatata 5100 taacattcaa
ataaaaatga aaataagaac tttcaaaaaa cagaactatg tttaatgtgt 5160
aaagattagt cgcacatcaa gtcatctgtt acaatatgtt acaacaagtc ataagcccaa
5220 caaagttagc acgtctaaat aaactaaaga gtccacgaaa atattacaaa
tcataagccc 5280 aacaaagtta ttgatcaaaa aaaaaaaacg cccaacaaag
ctaaacaaag tccaaaaaaa 5340 acttctcaag tctccatctt cctttatgaa
cattgaaaac tatacacaaa acaagtcaga 5400 taaatctctt tctgggcctg
tcttcccaac ctcctacatc acttccctat cggattgaat 5460 gttttacttg
taccttttcc gttgcaatga tattgatagt atgtttgtga aaactaatag 5520
ggttaacaat cgaagtcatg gaatatggat ttggtccaag attttccgag agctttctag
5580 tagaaagccc atcaccagaa atttactagt aaaataaatc accaattagg
tttcttatta 5640 tgtgccaaat tcaatataat tatagaggat atttcaaatg
aaaacgtatg aatgttatta 5700 gtaaatggtc aggtaagaca ttaaaaaaat
cctacgtcag atattcaact ttaaaaattc 5760 gatcagtgtg gaattgtaca
aaaatttggg atctactata tatatataat gctttacaac 5820 acttggattt
ttttttggag gctggaattt ttaatctaca tatttgtttt ggccatgcac 5880
caactcattg tttagtgtaa tactttgatt ttgtcaaata tatgtgttcg tgtatatttg
5940 tataagaatt tctttgacca tatacacaca cacatatata tatatatata
tatattatat 6000 atcatgcact tttaattgaa aaaataatat atatatatat
agtgcatttt ttctaacaac 6060 catatatgtt gcgattgatc tgcaaaaata
ctgctagagt aatgaaaaat ataatctatt 6120 gctgaaatta tctcagatgt
taagattttc ttaaagtaaa ttctttcaaa ttttagctaa 6180 aagtcttgta
ataactaaag aataatacac aatctcgacc acggaaaaaa aacacataat 6240
aaatttgaat ttcgaccgcg gtacccggaa ttgggttata attacctcag gtcgaggaat
6300 taattcggta cgtacctaat aacttcgtat agcatacatt atacgaagtt
atatggatct 6360 cgaggcatta cggcattacg gcactcgcga gggtcccaat
tcgagcatgg agccatttac 6420 aattgaatat atcctgccgc cgctgccgct
ttgcacccgg tggagcttgc atgttggttt 6480 ctacgcagaa ctgagccggt
taggcagata atttccattg agaactgagc catgtgcacc 6540 ttccccccaa
cacggtgagc gacggggcaa cggagtgatc cacatgggac ttttaaacat 6600
catccgtcgg atggcgttgc gagagaagca gtcgatccgt gagatcagcc gacgcaccgg
6660 gcaggcgcgc aacacgatcg caaagtattt gaacgcaggt acaatcgagc
cgacgttcac 6720 ggtaccggaa cgaccaagca agctagctta gtaaagccct
cgctagattt taatgcggat 6780 gttgcgatta cttcgccaac tattgcgata
acaagaaaaa gccagccttt catgatatat 6840 ctcccaattt gtgtagggct
tattatgcac gcttaaaaat aataaaagca gacttgacct 6900 gatagtttgg
ctgtgagcaa ttatgtgctt agtgcatcta acgcttgagt taagccgcgc 6960
cgcgaagcgg cgtcggcttg aacgaattgt tagacattat ttgccgacta ccttggtgat
7020 ctcgcctttc acgtagtgga caaattcttc caactgatct gcgcgcgagg
ccaagcgatc 7080 ttcttcttgt ccaagataag cctgtctagc ttcaagtatg
acgggctgat actgggccgg 7140 caggcgctcc attgcccagt cggcagcgac
atccttcggc gcgattttgc cggttactgc 7200 gctgtaccaa atgcgggaca
acgtaagcac tacatttcgc tcatcgccag cccagtcggg 7260 cggcgagttc
catagcgtta aggtttcatt tagcgcctca aatagatcct gttcaggaac 7320
cggatcaaag agttcctccg ccgctggacc taccaaggca acgctatgtt ctcttgcttt
7380 tgtcagcaag atagccagat caatgtcgat cgtggctggc tcgaagatac
ctgcaagaat 7440 gtcattgcgc tgccattctc caaattgcag ttcgcgctta
gctggataac gccacggaat 7500 gatgtcgtcg tgcacaacaa tggtgacttc
tacagcgcgg agaatctcgc tctctccagg 7560 ggaagccgaa gtttccaaaa
ggtcgttgat caaagctcgc cgcgttgttt catcaagcct 7620 tacggtcacc
gtaaccagca aatcaatatc actgtgtggc ttcaggccgc catccactgc 7680
ggagccgtac aaatgtacgg ccagcaacgt cggttcgaga tggcgctcga tgacgccaac
7740 tacctctgat
agttgagtcg atacttcggc gatcaccgct tccctcatga tgtttaactt 7800
tgttttaggg cgactgccct gctgcgtaac atcgttgctg ctccataaca tcaaacatcg
7860 acccacggcg taacgcgctt gctgcttgga tgcccgaggc atagactgta
ccccaaaaaa 7920 acagtcataa caagccatga aaaccgccac tgcgccgtta
ccaccgctgc gttcggtcaa 7980 ggttctggac cagttgcgtg agcgcatacg
ctacttgcat tacagcttac gaaccgaaca 8040 ggcttatgtc cactgggttc
gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac 8100 cttgggcagc
agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc 8160
ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca agtgctgtgc
8220 acggatctgc cctggcttca ggagatcgga agacctcggc cgtccgggcg
cttgccggtg 8280 gtgctgaccc cggatgaagt ctctagagct ctagagggtt
cgcatcctcg gttttctgga 8340 aggcgagcat cgtttgttcg cccagcttct
gtatggaacg ggcatgcgga tcagtgaggg 8400 tttgcaactg cgggtcaagg
atctggattt cgatcacggc acgatcatcg tgcgggaggg 8460 caagggctcc
aaggatcggg ccttgatgtt acccgagagc ttggcaccca gcctgcgcga 8520
gcagggatcg atccaacccc tccgctgcta tagtgcagtc ggcttctgac gttcagtgca
8580 gccgtcttct gaaaacgaca tgtcgcacaa gtcctaagtt acgcgacagg
ctgccgccct 8640 gcccttttcc tggcgttttc ttgtcgcgtg ttttagtcgc
ataaagtaga atacttgcga 8700 ctagaaccgg agacattacg ccatgaacaa
gagcgccgcc gctggcctgc tgggctatgc 8760 ccgcgtcagc accgacgacc
aggacttgac caaccaacgg gccgaactgc acgcggccgg 8820 ctgcaccaag
ctgttttccg agaagatcac cggcaccagg cgcgaccgcc cggagctggc 8880
caggatgctt gaccacctac gccctggcga cgttgtgaca gtgaccaggc tagaccgcct
8940 ggcccgcagc acccgcgacc tactggacat tgccgagcgc atccaggagg
ccggcgcggg 9000 cctgcgtagc ctggcagagc cgtgggccga caccaccacg
ccggccggcc gcatggtgtt 9060 gaccgtgttc gccggcattg ccgagttcga
gcgttcccta atcatcgacc gcacccggag 9120 cgggcgcgag gccgccaagg
cccgaggcgt gaagtttggc ccccgcccta ccctcacccc 9180 ggcacagatc
gcgcacgccc gcgagctgat cgaccaggaa ggccgcaccg tgaaagaggc 9240
ggctgcactg cttggcgtgc atcgctcgac cctgtaccgc gcacttgagc gcagcgagga
9300 agtgacgccc accgaggcca ggcggcgcgg tgccttccgt gaggacgcat
tgaccgaggc 9360 cgacgccctg gcggccgccg agaatgaacg ccaagaggaa
caagcatgaa accgcaccag 9420 gacggccagg acgaaccgtt tttcattacc
gaagagatcg aggcggagat gatcgcggcc 9480 gggtacgtgt tcgagccgcc
cgcgcacgtc tcaaccgtgc ggctgcatga aatcctggcc 9540 ggtttgtctg
atgccaagct ggcggcctgg ccggccagct tggccgctga agaaaccgag 9600
cgccgccgtc taaaaaggtg atgtgtattt gagtaaaaca gcttgcgtca tgcggtcgct
9660 gcgtatatga tgcgatgagt aaataaacaa atacgcaagg ggaacgcatg
aaggttatcg 9720 ctgtacttaa ccagaaaggc gggtcaggca agacgaccat
cgcaacccat ctagcccgcg 9780 ccctgcaact cgccggggcc gatgttctgt
tagtcgattc cgatccccag ggcagtgccc 9840 gcgattgggc ggccgtgcgg
gaagatcaac cgctaaccgt tgtcggcatc gaccgcccga 9900 cgattgaccg
cgacgtgaag gccatcggcc ggcgcgactt cgtagtgatc gacggagcgc 9960
cccaggcggc ggacttggct gtgtccgcga tcaaggcagc cgacttcgtg ctgattccgg
10020 tgcagccaag cccttacgac atatgggcca ccgccgacct ggtggagctg
gttaagcagc 10080 gcattgaggt cacggatgga aggctacaag cggcctttgt
cgtgtcgcgg gcgatcaaag 10140 gcacgcgcat cggcggtgag gttgccgagg
cgctggccgg gtacgagctg cccattcttg 10200 agtcccgtat cacgcagcgc
gtgagctacc caggcactgc cgccgccggc acaaccgttc 10260 ttgaatcaga
acccgagggc gacgctgccc gcgaggtcca ggcgctggcc gctgaaatta 10320
aatcaaaact catttgagtt aatgaggtaa agagaaaatg agcaaaagca caaacacgct
10380 aagtgccggc cgtccgagcg cacgcagcag caaggctgca acgttggcca
gcctggcaga 10440 cacgccagcc atgaagcggg tcaactttca gttgccggcg
gaggatcaca ccaagctgaa 10500 gatgtacgcg gtacgccaag gcaagaccat
taccgagctg ctatctgaat acatcgcgca 10560 gctaccagag taaatgagca
aatgaataaa tgagtagatg aattttagcg gctaaaggag 10620 gcggcatgga
aaatcaagaa caaccaggca ccgacgccgt ggaatgcccc atgtgtggag 10680
gaacgggcgg ttggccaggc gtaagcggct gggttgtctg ccggccctgc aatggcactg
10740 gaacccccaa gcccgaggaa tcggcgtgac ggtcgcaaac catccggccc
ggtacaaatc 10800 ggcgcggcgc tgggtgatga cctggtggag aagttgaagg
ccgcgcaggc cgcccagcgg 10860 caacgcatcg aggcagaagc acgccccggt
gaatcgtggc aagcggccgc tgatcgaatc 10920 cgcaaagaat cccggcaacc
gccggcagcc ggtgcgccgt cgattaggaa gccgcccaag 10980 ggcgacgagc
aaccagattt tttcgttccg atgctctatg acgtgggcac ccgcgatagt 11040
cgcagcatca tggacgtggc cgttttccgt ctgtcgaagc gtgaccgacg agctggcgag
11100 gtgatccgct acgagcttcc agacgggcac gtagaggttt ccgcagggcc
ggccggcatg 11160 gccagtgtgt gggattacga cctggtactg atggcggttt
cccatctaac cgaatccatg 11220 aaccgatacc gggaagggaa gggagacaag
cccggccgcg tgttccgtcc acacgttgcg 11280 gacgtactca agttctgccg
gcgagccgat ggcggaaagc agaaagacga cctggtagaa 11340 acctgcattc
ggttaaacac cacgcacgtt gccatgcagc gtacgaagaa ggccaagaac 11400
ggccgcctgg tgacggtatc cgagggtgaa gccttgatta gccgctacaa gatcgtaaag
11460 agcgaaaccg ggcggccgga gtacatcgag atcgagctag ctgattggat
gtaccgcgag 11520 atcacagaag gcaagaaccc ggacgtgctg acggttcacc
ccgattactt tttgatcgat 11580 cccggcatcg gccgttttct ctaccgcctg
gcacgccgcg ccgcaggcaa ggcagaagcc 11640 agatggttgt tcaagacgat
ctacgaacgc agtggcagcg ccggagagtt caagaagttc 11700 tgtttcaccg
tgcgcaagct gatcgggtca aatgacctgc cggagtacga tttgaaggag 11760
gaggcggggc aggctggccc gatcctagtc atgcgctacc gcaacctgat cgagggcgaa
11820 gcatccgccg gttcctaatg tacggagcag atgctagggc aaattgccct
agcaggggaa 11880 aaaggtcgaa aaggtctctt tcctgtggat agcacgtaca
ttgggaaccc aaagccgtac 11940 attgggaacc ggaacccgta cattgggaac
ccaaagccgt acattgggaa ccggtcacac 12000 atgtaagtga ctgatataaa
agagaaaaaa ggcgattttt ccgcctaaaa ctctttaaaa 12060 cttattaaaa
ctcttaaaac ccgcctggcc tgtgcataac tgtctggcca gcgcacagcc 12120
gaagagctgc aaaaagcgcc tacccttcgg tcgctgcgct ccctacgccc cgccgcttcg
12180 cgtcggccta tcgcggccgc tggccgctca aaaatggctg gcctacggcc
aggcaatcta 12240 ccagggcgcg gacaagccgc gccgtcgcca ctcgaccgcc
ggcgcccaca tcaaggcacc 12300 ctgcctcgcg cgtttcggtg atgacggtga
aaacctctga cacatgcagc tcccggagac 12360 ggtcacagct tgtctgtaag
cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 12420 gggtgttggc
gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta 12480
tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
12540 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
cgcttcctcg 12600 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
cggtatcagc tcactcaaag 12660 gcggtaatac ggttatccac agaatcaggg
gataacgcag gaaagaacat gtgagcaaaa 12720 ggccagcaaa aggccaggaa
ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 12780 cgcccccctg
acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 12840
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
12900 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
ggcgctttct 12960 catagctcac gctgtaggta tctcagttcg gtgtaggtcg
ttcgctccaa gctgggctgt 13020 gtgcacgaac cccccgttca gcccgaccgc
tgcgccttat ccggtaacta tcgtcttgag 13080 tccaacccgg taagacacga
cttatcgcca ctggcagcag ccactggtaa caggattagc 13140 agagcgaggt
atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 13200
actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
13260 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
ttttgtttgc 13320 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
atccggaaaa cgcaagcgca 13380 aagagaaagc aggtagcttg cagtgggctt
acatggcgat agctagactg ggcggtttta 13440 tggacagcaa gcgaaccgga attgcc
13466 10 31 PRT Artificial Sequence Consensus sequence 1 of PARG
protein 10 Leu Xaa Val Asp Phe Ala Asn Xaa Xaa Xaa Gly Gly Gly Xaa
Xaa Xaa 1 5 10 15 Xaa Gly Xaa Val Gln Glu Glu Ile Arg Phe Xaa Xaa
Xaa Pro Glu 20 25 30 11 20 PRT Artificial Sequence Consensus
sequence 2 for PARG protein 11 Thr Gly Xaa Trp Gly Cys Gly Ala Phe
Xaa Gly Asp Xaa Xaa Leu Lys 1 5 10 15 Xaa Xaa Xaa Gln 20 12 13 PRT
Artificial Sequence Consensus sequence 3 for PARG protein 12 Asp
Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Ala Ile Asp Ala 1 5 10 13 10 PRT
Artificial Sequence Consensus sequence 4 for PARG protein 13 Arg
Glu Xaa Xaa Lys Ala Xaa Xaa Gly Phe 1 5 10 14 11 PRT Artificial
Sequence conserved PARG region 14 Gly Xaa Xaa Xaa Xaa Ser Xaa Tyr
Thr Gly Tyr 1 5 10 15 1530 DNA Oryza sativa CDS (1)..(1530) 15 atg
gag gcg cgc ggc gac ctg cgc tcg atc ctg ccc tac ctc ccc gtc 48 Met
Glu Ala Arg Gly Asp Leu Arg Ser Ile Leu Pro Tyr Leu Pro Val 1 5 10
15 gtg ctc cgc ggc ggc gcg ctc ttc tgg ccg ccg gcg gcg cag gag gcg
96 Val Leu Arg Gly Gly Ala Leu Phe Trp Pro Pro Ala Ala Gln Glu Ala
20 25 30 ctc aag gcg ctg gcg ctg ggc ccc gac gtg agc cgc gtc tcc
tcc ggc 144 Leu Lys Ala Leu Ala Leu Gly Pro Asp Val Ser Arg Val Ser
Ser Gly 35 40 45 gac gtc ctc gcc gac gcc ctc acc gac ctc cgc ctc
gcg ctc aac ctc 192 Asp Val Leu Ala Asp Ala Leu Thr Asp Leu Arg Leu
Ala Leu Asn Leu 50 55 60 gac cca ctc ccg cgc cgc gcc gcc gag ggc
ttc gcg ctc ttc ttc gac 240 Asp Pro Leu Pro Arg Arg Ala Ala Glu Gly
Phe Ala Leu Phe Phe Asp 65 70 75 80 gac ctc ctg tcg cgg gcg cag gcg
cgg gac tgg ttc gac cac gtc gcc 288 Asp Leu Leu Ser Arg Ala Gln Ala
Arg Asp Trp Phe Asp His Val Ala 85 90 95 ccc tcc ctc gcc cgc ctc
ctc ctc cgc ctc ccc acg ctg ctc gag ggc 336 Pro Ser Leu Ala Arg Leu
Leu Leu Arg Leu Pro Thr Leu Leu Glu Gly 100 105 110 cac tac cgc gcc
gcc ggc gac gag gct cgc ggg ctc cgc atc ctg agc 384 His Tyr Arg Ala
Ala Gly Asp Glu Ala Arg Gly Leu Arg Ile Leu Ser 115 120 125 tcg cag
gat gcc ggg ctc gtg ctc ctc agc cag gag ctc gcc gcc gcg 432 Ser Gln
Asp Ala Gly Leu Val Leu Leu Ser Gln Glu Leu Ala Ala Ala 130 135 140
ctg ctc gcc tgc gcg ctc ttc tgc ctg ttc ccc acc gcc gat agg gcc 480
Leu Leu Ala Cys Ala Leu Phe Cys Leu Phe Pro Thr Ala Asp Arg Ala 145
150 155 160 gag gcg tgc ctc ccg gcg atc aat ttc gat agc cta ttt gcg
gca ctg 528 Glu Ala Cys Leu Pro Ala Ile Asn Phe Asp Ser Leu Phe Ala
Ala Leu 165 170 175 tgt tat aat tcg agg caa agc cag gag cag aag gtg
agg tgc ctt gtt 576 Cys Tyr Asn Ser Arg Gln Ser Gln Glu Gln Lys Val
Arg Cys Leu Val 180 185 190 cac tat ttt gac agg gtg acc gct tct aca
cct act ggt tcc gtt tcg 624 His Tyr Phe Asp Arg Val Thr Ala Ser Thr
Pro Thr Gly Ser Val Ser 195 200 205 ttt gag cgt aag gtt ctt cct cgc
cgt cct gaa tct gat ggc att acg 672 Phe Glu Arg Lys Val Leu Pro Arg
Arg Pro Glu Ser Asp Gly Ile Thr 210 215 220 tac cct gac atg gat act
tgg atg aaa tct ggt gtt ccc ctt tgc aca 720 Tyr Pro Asp Met Asp Thr
Trp Met Lys Ser Gly Val Pro Leu Cys Thr 225 230 235 240 ttc cgg gta
ttt tcc tca ggc ttg ata gaa gat gag gaa caa gaa gcc 768 Phe Arg Val
Phe Ser Ser Gly Leu Ile Glu Asp Glu Glu Gln Glu Ala 245 250 255 ctt
gaa gtt gac ttt gca aat aga tat ttg gga ggt ggc gca ctt tcc 816 Leu
Glu Val Asp Phe Ala Asn Arg Tyr Leu Gly Gly Gly Ala Leu Ser 260 265
270 aga ggc tgc gtg cag gaa gaa atc cgg ttc atg ata aac cca gaa ttg
864 Arg Gly Cys Val Gln Glu Glu Ile Arg Phe Met Ile Asn Pro Glu Leu
275 280 285 atc gtg ggc atg ctc ttc atg gtt tca atg gaa gat aat gaa
gct ata 912 Ile Val Gly Met Leu Phe Met Val Ser Met Glu Asp Asn Glu
Ala Ile 290 295 300 gaa att gtt ggt gca gaa agg ttc tca cag tac atg
ggg tat ggt tcc 960 Glu Ile Val Gly Ala Glu Arg Phe Ser Gln Tyr Met
Gly Tyr Gly Ser 305 310 315 320 tca ttc cgt ttt act ggt gac tac tta
gat agc aaa ccc ttt gat gcg 1008 Ser Phe Arg Phe Thr Gly Asp Tyr
Leu Asp Ser Lys Pro Phe Asp Ala 325 330 335 atg ggt aga cgg aaa act
agg ata gtg gca att gat gct ttg gac tgt 1056 Met Gly Arg Arg Lys
Thr Arg Ile Val Ala Ile Asp Ala Leu Asp Cys 340 345 350 cca act agg
tta cag ttt gaa tct agt ggt ctt cta agg gaa gtg aac 1104 Pro Thr
Arg Leu Gln Phe Glu Ser Ser Gly Leu Leu Arg Glu Val Asn 355 360 365
aag gct ttt tgt gga ttt ttg gat caa tca aat cat cag ctc tgt gca
1152 Lys Ala Phe Cys Gly Phe Leu Asp Gln Ser Asn His Gln Leu Cys
Ala 370 375 380 aag ctt gtc cag gat tta aat aca aag gat aac tgt cca
agt gtc att 1200 Lys Leu Val Gln Asp Leu Asn Thr Lys Asp Asn Cys
Pro Ser Val Ile 385 390 395 400 cct gat gaa tgc ata gga gtt tca act
gga aac tgg ggt tgc ggg gct 1248 Pro Asp Glu Cys Ile Gly Val Ser
Thr Gly Asn Trp Gly Cys Gly Ala 405 410 415 ttt ggt gga aac cct gaa
atc aag agc atg att caa tgg att gct gca 1296 Phe Gly Gly Asn Pro
Glu Ile Lys Ser Met Ile Gln Trp Ile Ala Ala 420 425 430 tca cag gca
ctc cga tct ttt att aac tac tac act ttt gag tcc gaa 1344 Ser Gln
Ala Leu Arg Ser Phe Ile Asn Tyr Tyr Thr Phe Glu Ser Glu 435 440 445
tca ctg aaa aga tta gaa gag gtg acc cag tgg ata ttg cgc cat agg
1392 Ser Leu Lys Arg Leu Glu Glu Val Thr Gln Trp Ile Leu Arg His
Arg 450 455 460 tgg acg gtt ggc gag ttg tgg gac atg ctt gtg gag tat
tca tcc cag 1440 Trp Thr Val Gly Glu Leu Trp Asp Met Leu Val Glu
Tyr Ser Ser Gln 465 470 475 480 agg cta aga gga gac acc aat gag ggc
ttt tta aca tgg cta ctt ccc 1488 Arg Leu Arg Gly Asp Thr Asn Glu
Gly Phe Leu Thr Trp Leu Leu Pro 485 490 495 aag gac atc ccc aat ggt
gat gta gat tac atg tgt gaa tag 1530 Lys Asp Ile Pro Asn Gly Asp
Val Asp Tyr Met Cys Glu 500 505 16 509 PRT Oryza sativa 16 Met Glu
Ala Arg Gly Asp Leu Arg Ser Ile Leu Pro Tyr Leu Pro Val 1 5 10 15
Val Leu Arg Gly Gly Ala Leu Phe Trp Pro Pro Ala Ala Gln Glu Ala 20
25 30 Leu Lys Ala Leu Ala Leu Gly Pro Asp Val Ser Arg Val Ser Ser
Gly 35 40 45 Asp Val Leu Ala Asp Ala Leu Thr Asp Leu Arg Leu Ala
Leu Asn Leu 50 55 60 Asp Pro Leu Pro Arg Arg Ala Ala Glu Gly Phe
Ala Leu Phe Phe Asp 65 70 75 80 Asp Leu Leu Ser Arg Ala Gln Ala Arg
Asp Trp Phe Asp His Val Ala 85 90 95 Pro Ser Leu Ala Arg Leu Leu
Leu Arg Leu Pro Thr Leu Leu Glu Gly 100 105 110 His Tyr Arg Ala Ala
Gly Asp Glu Ala Arg Gly Leu Arg Ile Leu Ser 115 120 125 Ser Gln Asp
Ala Gly Leu Val Leu Leu Ser Gln Glu Leu Ala Ala Ala 130 135 140 Leu
Leu Ala Cys Ala Leu Phe Cys Leu Phe Pro Thr Ala Asp Arg Ala 145 150
155 160 Glu Ala Cys Leu Pro Ala Ile Asn Phe Asp Ser Leu Phe Ala Ala
Leu 165 170 175 Cys Tyr Asn Ser Arg Gln Ser Gln Glu Gln Lys Val Arg
Cys Leu Val 180 185 190 His Tyr Phe Asp Arg Val Thr Ala Ser Thr Pro
Thr Gly Ser Val Ser 195 200 205 Phe Glu Arg Lys Val Leu Pro Arg Arg
Pro Glu Ser Asp Gly Ile Thr 210 215 220 Tyr Pro Asp Met Asp Thr Trp
Met Lys Ser Gly Val Pro Leu Cys Thr 225 230 235 240 Phe Arg Val Phe
Ser Ser Gly Leu Ile Glu Asp Glu Glu Gln Glu Ala 245 250 255 Leu Glu
Val Asp Phe Ala Asn Arg Tyr Leu Gly Gly Gly Ala Leu Ser 260 265 270
Arg Gly Cys Val Gln Glu Glu Ile Arg Phe Met Ile Asn Pro Glu Leu 275
280 285 Ile Val Gly Met Leu Phe Met Val Ser Met Glu Asp Asn Glu Ala
Ile 290 295 300 Glu Ile Val Gly Ala Glu Arg Phe Ser Gln Tyr Met Gly
Tyr Gly Ser 305 310 315 320 Ser Phe Arg Phe Thr Gly Asp Tyr Leu Asp
Ser Lys Pro Phe Asp Ala 325 330 335 Met Gly Arg Arg Lys Thr Arg Ile
Val Ala Ile Asp Ala Leu Asp Cys 340 345 350 Pro Thr Arg Leu Gln Phe
Glu Ser Ser Gly Leu Leu Arg Glu Val Asn 355 360 365 Lys Ala Phe Cys
Gly Phe Leu Asp Gln Ser Asn His Gln Leu Cys Ala 370 375 380 Lys Leu
Val Gln Asp Leu Asn Thr Lys Asp Asn Cys Pro Ser Val Ile 385 390 395
400 Pro Asp Glu Cys Ile Gly Val Ser Thr Gly Asn Trp Gly Cys Gly Ala
405 410 415 Phe Gly Gly Asn Pro Glu Ile Lys Ser Met Ile Gln Trp Ile
Ala Ala 420 425 430 Ser Gln Ala Leu Arg Ser Phe Ile Asn Tyr Tyr Thr
Phe Glu Ser Glu 435 440 445 Ser Leu Lys Arg Leu Glu Glu Val Thr Gln
Trp Ile Leu Arg His Arg 450 455 460 Trp Thr Val Gly Glu Leu Trp Asp
Met Leu Val Glu Tyr Ser Ser Gln 465 470 475 480 Arg Leu Arg Gly Asp
Thr Asn Glu Gly Phe Leu Thr Trp Leu Leu Pro 485 490 495 Lys Asp Ile
Pro Asn Gly Asp Val Asp Tyr Met Cys Glu 500 505 17 25 DNA
Artificial Sequence degenerate oligonucleotide primer PG1 17
atgtbccaca rmtckccrac mgtcc 25 18 28 DNA Artificial Sequence
degenerate oligonucleotide primer PG2 18 gggtytccwc caaaarcmcc
rcawcccc 28 19 26 DNA
Artificial Sequence degenerate oligonucleotide primer PG3 19
gctatagaaa twgtyggtgy rgaaag 26 20 26 DNA Artificial Sequence
degenerate oligonucleotide primer PG4 20 agrggstgyg trcaggarga
ratmcg 26 21 23 DNA Artificial Sequence degenerate oligonucleotide
primer PG5 21 atggargaya aygargcnat hga 23 22 24 DNA Artificial
Sequence degenerate oligonucleotide primer PG6 22 ccaytgdagc
atrctyttda gytc 24 23 603 DNA zea mays 23 tagggctgtg tgcaggagga
aatccgcttc atgataaacc ccgaattgat tgtgggtatg 60 ctattcttgt
cttgtatgga agataacgag gctatagaaa tctttggtgc agaacggttc 120
tcacagtata tgggttatgg ttcctccttt cgctttgttg gtgactattt agataccaaa
180 ccctttgatt cgatgggcag acggagaact aggattgtgg ctatcgatgc
tttggactgt 240 ccagctaggt tacactatga atctggctgt ctcctaaggg
aagtgaacaa ggcattttgt 300 ggatttttcg atcaatcgaa acaccatctc
tatgcgaagc ttttccagga tttgcacaac 360 aaggatgact tttcaagcat
caattccagt gagtacgtag gagtttcaac aggaaactgg 420 ggttgtggtg
cttttggtgg aaaccctgaa atcaagagca tgattcagtg gattgctgca 480
tcacaggctc ttcgcccttt tgttaattac tacacttttg agaacgtgtc tctgcaaaga
540 ttagaggagg tgatccagtg gatacggctt catggctgga ctgtcggcga
gctgtggaac 600 ata 603 24 12987 DNA Artificial sequence T-DNA
vector comprising a chimeric ParG expression reducing gene 24
cggcaggata tattcaattg taaatggctc catggcgatc gctctagagg atcttcccga
60 tctagtaaca tagatgacac cgcgcgcgat aatttatcct agtttgcgcg
ctatattttg 120 ttttctatcg cgtattaaat gtataattgc gggactctaa
tcataaaaac ccatctcata 180 aataacgtca tgcattacat gttaattatt
acatgcttaa cgtaattcaa cagaaattat 240 atgataatca tcgcaagacc
ggcaacagga ttcaatctta agaaacttta ttgccaaatg 300 tttgaacgat
ctgcttcgga tcctagacgc gtgagatcag atctcggtga cgggcaggac 360
cggacggggc ggtaccggca ggctgaagtc cagctgccag aaacccacgt catgccagtt
420 cccgtgcttg aagccggccg cccgcagcat gccgcggggg gcatatccga
gcgcctcgtg 480 catgcgcacg ctcgggtcgt tgggcagccc gatgacagcg
accacgctct tgaagccctg 540 tgcctccagg gacttcagca ggtgggtgta
gagcgtggag cccagtcccg tccgctggtg 600 gcggggggag acgtacacgg
tcgactcggc cgtccagtcg taggcgttgc gtgccttcca 660 ggggcccgcg
taggcgatgc cggcgacctc gccgtccacc tcggcgacga gccagggata 720
gcgctcccgc agacggacga ggtcgtccgt ccactcctgc ggttcctgcg gctcggtacg
780 gaagttgacc gtgcttgtct cgatgtagtg gttgacgatg gtgcagaccg
ccggcatgtc 840 cgcctcggtg gcacggcgga tgtcggccgg gcgtcgttct
gggtccatgg ttatagagag 900 agagatagat ttatagagag agactggtga
tttcagcgtg tcctctccaa atgaaatgaa 960 cttccttata tagaggaagg
gtcttgcgaa ggatagtggg attgtgcgtc atcccttacg 1020 tcagtggaga
tgtcacatca atccacttgc tttgaagacg tggttggaac gtcttctttt 1080
tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc agaggcatct
1140 tgaatgatag cctttccttt atcgcaatga tggcatttgt aggagccacc
ttccttttct 1200 actgtccttt cgatgaagtg acagatagct gggcaatgga
atccgaggag gtttcccgaa 1260 attatccttt gttgaaaagt ctcaatagcc
ctttggtctt ctgagactgt atctttgaca 1320 tttttggagt agaccagagt
gtcgtgctcc accatgttga cgaagatttt cttcttgtca 1380 ttgagtcgta
aaagactctg tatgaactgt tcgccagtct tcacggcgag ttctgttaga 1440
tcctcgattt gaatcttaga ctccatgcat ggccttagat tcagtaggaa ctaccttttt
1500 agagactcca atctctatta cttgccttgg tttatgaagc aagccttgaa
tcgtccatac 1560 tggaatagta cttctgatct tgagaaatat gtctttctct
gtgttcttga tgcaattagt 1620 cctgaatctt ttgactgcat ctttaacctt
cttgggaagg tatttgatct cctggagatt 1680 gttactcggg tagatcgtct
tgatgagacc tgctgcgtag gaacgcggcc gcgtatacgt 1740 atcgatatct
tcgaattcga gctcgtcgag cggccgctcg acgaattaat tccaatccca 1800
caaaaatctg agcttaacag cacagttgct cctctcagag cagaatcggg tattcaacac
1860 cctcatatca actactacgt tgtgtataac ggtccacatg ccggtatata
cgatgactgg 1920 ggttgtacaa aggcggcaac aaacggcgtt cccggagttg
cacacaagaa atttgccact 1980 attacagagg caagagcagc agctgacgcg
tacacaacaa gtcagcaaac agacaggttg 2040 aacttcatcc ccaaaggaga
agctcaactc aagcccaaga gctttgctaa ggccctaaca 2100 agcccaccaa
agcaaaaagc ccactggctc acgctaggaa ccaaaaggcc cagcagtgat 2160
ccagccccaa aagagatctc ctttgccccg gagattacaa tggacgattt cctctatctt
2220 tacgatctag gaaggaagtt cgaaggtgaa ggtgacgaca ctatgttcac
cactgataat 2280 gagaaggtta gcctcttcaa tttcagaaag aatgctgacc
cacagatggt tagagaggcc 2340 tacgcagcag gtctcatcaa gacgatctac
ccgagtaaca atctccagga gatcaaatac 2400 cttcccaaga aggttaaaga
tgcagtcaaa agattcagga ctaattgcat caagaacaca 2460 gagaaagaca
tatttctcaa gatcagaagt actattccag tatggacgat tcaaggcttg 2520
cttcataaac caaggcaagt aatagagatt ggagtctcta aaaaggtagt tcctactgaa
2580 tctaaggcca tgcatggagt ctaagattca aatcgaggat ctaacagaac
tcgccgtgaa 2640 gactggcgaa cagttcatac agagtctttt acgactcaat
gacaagaaga aaatcttcgt 2700 caacatggtg gagcacgaca ctctggtcta
ctccaaaaat gtcaaagata cagtctcaga 2760 agaccaaagg gctattgaga
cttttcaaca aaggataatt tcgggaaacc tcctcggatt 2820 ccattgccca
gctatctgtc acttcatcga aaggacagta gaaaaggaag gtggctccta 2880
caaatgccat cattgcgata aaggaaaggc tatcattcaa gatctctctg ccgacagtgg
2940 tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg
ttccaaccac 3000 gtcttcaaag caagtggatt gatgtgacat ctccactgac
gtaagggatg acgcacaatc 3060 ccactatcct tcgcaagacc cttcctctat
ataaggaagt tcatttcatt tggagaggac 3120 acgctcgagc ccgaattgat
tgtgggtatg ctattcttgt cttgtatgga agataacgag 3180 gctatagaaa
tctttggtgc agaacggttc tcacagtata tgggttatgg ttcctccttt 3240
cgctttgttg gtgactattt agataccaaa ccctttgatt cgatgggcag acggagaact
3300 aggattgtgg cggtacccca gcttggtaag gaaataatta ttttcttttt
tccttttagt 3360 ataaaatagt taagtgatgt taattagtat gattataata
atatagttgt tataattgtg 3420 aaaaaataat ttataaatat attgtttaca
taaacaacat agtaatgtaa aaaaatatga 3480 caagtgatgt gtaagacgaa
gaagataaaa gttgagagta agtatattat ttttaatgaa 3540 tttgatcgaa
catgtaagat gatatactag cattaatatt tgttttaatc ataatagtaa 3600
ttctagctgg tttgatgaat taaatatcaa tgataaaata ctatagtaaa aataagaata
3660 aataaattaa aataatattt ttttatgatt aatagtttat tatataatta
aatatctata 3720 ccattactaa atattttagt ttaaaagtta ataaatattt
tgttagaaat tccaatctgc 3780 ttgtaattta tcaataaaca aaatattaaa
taacaagcta aagtaacaaa taatatcaaa 3840 ctaatagaaa cagtaatcta
atgtaacaaa acataatcta atgctaatat aacaaagcgc 3900 aagatctatc
attttatata gtattatttt caatcaacat tcttattaat ttctaaataa 3960
tacttgtagt tttattaact tctaaatgga ttgactatta attaaatgaa ttagtcgaac
4020 atgaataaac aaggtaacat gatagatcat gtcattgtgt tatcattgat
cttacatttg 4080 gattgattac agttgggaag ctgggttcga aatcgatagc
cacaatccta gttctccgtc 4140 tgcccatcga atcaaagggt ttggtatcta
aatagtcacc aacaaagcga aaggaggaac 4200 cataacccat atactgtgag
aaccgttctg caccaaagat ttctatagcc tcgttatctt 4260 ccatacaaga
caagaatagc atacccacaa tcaattcggg tctagagtcc tgctttaatg 4320
agatatgcga gacgcctatg atcgcatgat atttgctttc aattctgttg tgcacgttgt
4380 aaaaaacctg agcatgtgta gctcagatcc ttaccgccgg tttcggttca
ttctaatgaa 4440 tatatcaccc gttactatcg tatttttatg aataatattc
tccgttcaat ttactgattg 4500 taccctacta cttatatgta caatattaaa
atgaaaacaa tatattgtgc tgaataggtt 4560 tatagcgaca tctatgatag
agcgccacaa taacaaacaa ttgcgtttta ttattacaaa 4620 tccaatttta
aaaaaagcgg cagaaccggt caaacctaaa agactgatta cataaatctt 4680
attcaaattt caaaaggccc caggggctag tatctacgac acaccgagcg gcgaactaat
4740 aacgttcact gaagggaact ccggttcccc gccggcgcgc atgggtgaga
ttccttgaag 4800 ttgagtattg gccgtccgct ctaccgaaag ttacgggcac
cattcaaccc ggtccagcac 4860 ggcggccggg taaccgactt gctgccccga
gaattatgca gcattttttt ggtgtatgtg 4920 ggccccaaat gaagtgcagg
tcaaaccttg acagtgacga caaatcgttg ggcgggtcca 4980 gggcgaattt
tgcgacaaca tgtcgaggct cagcaggacc tgcaggtcga cggccgagta 5040
ctggcaggat atataccgtt gtaatttgtc gcgtgtgaat aagtcgctgt gtatgtttgt
5100 ttgattgttt ctgttggagt gcagcccatt tcaccggaca agtcggctag
attgatttag 5160 ccctgatgaa ctgccgaggg gaagccatct tgagcgcgga
atgggaatgg atttcgttgt 5220 acaacgagac gacagaacac ccacgggacc
gagcttcgat cgagcatcaa atgaaactgc 5280 aatttattca tatcaggatt
atcaatacca tatttttgaa aaagccgttt ctgtaatgaa 5340 ggagaaaact
caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt 5400
ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
5460 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
tttatgcatt 5520 tctttccaga cttgttcaac aggccagcca ttacgctcgt
catcaaaatc actcgcatca 5580 accaaaccgt tattcattcg tgattgcgcc
tgagcgagac gaaatacgcc gctgttaaaa 5640 ggacaattac aaacaggaat
cgaatgcaac cggcgcagga acactgccag cgcatcaaca 5700 atattttcac
ctgaatcagg atattcttct aatacctgga atgctgtttt tccggggatc 5760
gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
5820 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
attggcaacg 5880 ctacctttgc catgtttcag aaacaactct ggcgcatcgg
gcttcccata caatcgatag 5940 attgtcgcac ctgattgccc gacattatcc
gaatctggca attccggttc gcttgctgtc 6000 cataaaaccg cccagtctag
ctatcgccat gtaagcccac tgcaagctac ctgctttctc 6060 tttgcgcttg
cgttttccgg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 6120
ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
6180 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
tgtccttcta 6240 gtgtagccgt agttaggcca ccacttcaag aactctgtag
caccgcctac atacctcgct 6300 ctgctaatcc tgttaccagt ggctgctgcc
agtggcgata agtcgtgtct taccgggttg 6360 gactcaagac gatagttacc
ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 6420 acacagccca
gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 6480
tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
6540 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
tctttatagt 6600 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
tgtgatgctc gtcagggggg 6660 cggagcctat ggaaaaacgc cagcaacgcg
gcctttttac ggttcctggc cttttgctgg 6720 ccttttgctc acatgttctt
tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 6780 gcctttgagt
gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 6840
agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
6900 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
gttaagccag 6960 tatacactcc gctatcgcta cgtgactggg tcatggctgc
gccccgacac ccgccaacac 7020 ccgctgacgc gccctgacgg gcttgtctgc
tcccggcatc cgcttacaga caagctgtga 7080 ccgtctccgg gagctgcatg
tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc 7140 agggtgcctt
gatgtgggcg ccggcggtcg agtggcgacg gcgcggcttg tccgcgccct 7200
ggtagattgc ctggccgtag gccagccatt tttgagcggc cagcggccgc gataggccga
7260 cgcgaagcgg cggggcgtag ggagcgcagc gaccgaaggg taggcgcttt
ttgcagctct 7320 tcggctgtgc gctggccaga cagttatgca caggccaggc
gggttttaag agttttaata 7380 agttttaaag agttttaggc ggaaaaatcg
ccttttttct cttttatatc agtcacttac 7440 atgtgtgacc ggttcccaat
gtacggcttt gggttcccaa tgtacgggtt ccggttccca 7500 atgtacggct
ttgggttccc aatgtacgtg ctatccacag gaaagagacc ttttcgacct 7560
ttttcccctg ctagggcaat ttgccctagc atctgctccg tacattagga accggcggat
7620 gcttcgccct cgatcaggtt gcggtagcgc atgactagga tcgggccagc
ctgccccgcc 7680 tcctccttca aatcgtactc cggcaggtca tttgacccga
tcagcttgcg cacggtgaaa 7740 cagaacttct tgaactctcc ggcgctgcca
ctgcgttcgt agatcgtctt gaacaaccat 7800 ctggcttctg ccttgcctgc
ggcgcggcgt gccaggcggt agagaaaacg gccgatgccg 7860 ggatcgatca
aaaagtaatc ggggtgaacc gtcagcacgt ccgggttctt gccttctgtg 7920
atctcgcggt acatccaatc agctagctcg atctcgatgt actccggccg cccggtttcg
7980 ctctttacga tcttgtagcg gctaatcaag gcttcaccct cggataccgt
caccaggcgg 8040 ccgttcttgg ccttcttcgt acgctgcatg gcaacgtgcg
tggtgtttaa ccgaatgcag 8100 gtttctacca ggtcgtcttt ctgctttccg
ccatcggctc gccggcagaa cttgagtacg 8160 tccgcaacgt gtggacggaa
cacgcggccg ggcttgtctc ccttcccttc ccggtatcgg 8220 ttcatggatt
cggttagatg ggaaaccgcc atcagtacca ggtcgtaatc ccacacactg 8280
gccatgccgg ccggccctgc ggaaacctct acgtgcccgt ctggaagctc gtagcggatc
8340 acctcgccag ctcgtcggtc acgcttcgac agacggaaaa cggccacgtc
catgatgctg 8400 cgactatcgc gggtgcccac gtcatagagc atcggaacga
aaaaatctgg ttgctcgtcg 8460 cccttgggcg gcttcctaat cgacggcgca
ccggctgccg gcggttgccg ggattctttg 8520 cggattcgat cagcggccgc
ttgccacgat tcaccggggc gtgcttctgc ctcgatgcgt 8580 tgccgctggg
cggcctgcgc ggccttcaac ttctccacca ggtcatcacc cagcgccgcg 8640
ccgatttgta ccgggccgga tggtttgcga ccgtcacgcc gattcctcgg gcttgggggt
8700 tccagtgcca ttgcagggcc ggcagacaac ccagccgctt acgcctggcc
aaccgcccgt 8760 tcctccacac atggggcatt ccacggcgtc ggtgcctggt
tgttcttgat tttccatgcc 8820 gcctccttta gccgctaaaa ttcatctact
catttattca tttgctcatt tactctggta 8880 gctgcgcgat gtattcagat
agcagctcgg taatggtctt gccttggcgt accgcgtaca 8940 tcttcagctt
ggtgtgatcc tccgccggca actgaaagtt gacccgcttc atggctggcg 9000
tgtctgccag gctggccaac gttgcagcct tgctgctgcg tgcgctcgga cggccggcac
9060 ttagcgtgtt tgtgcttttg ctcattttct ctttacctca ttaactcaaa
tgagttttga 9120 tttaatttca gcggccagcg cctggacctc gcgggcagcg
tcgccctcgg gttctgattc 9180 aagaacggtt gtgccggcgg cggcagtgcc
tgggtagctc acgcgctgcg tgatacggga 9240 ctcaagaatg ggcagctcgt
acccggccag cgcctcggca acctcaccgc cgatgcgcgt 9300 gcctttgatc
gcccgcgaca cgacaaaggc cgcttgtagc cttccatccg tgacctcaat 9360
gcgctgctta accagctcca ccaggtcggc ggtggcccat atgtcgtaag ggcttggctg
9420 caccggaatc agcacgaagt cggctgcctt gatcgcggac acagccaagt
ccgccgcctg 9480 gggcgctccg tcgatcacta cgaagtcgcg ccggccgatg
gccttcacgt cgcggtcaat 9540 cgtcgggcgg tcgatgccga caacggttag
cggttgatct tcccgcacgg ccgcccaatc 9600 gcgggcactg ccctggggat
cggaatcgac taacagaaca tcggccccgg cgagttgcag 9660 ggcgcgggct
agatgggttg cgatggtcgt cttgcctgac ccgcctttct ggttaagtac 9720
agcgataacc ttcatgcgtt ccccttgcgt atttgtttat ttactcatcg catcatatac
9780 gcagcgaccg catgacgcaa gctgttttac tcaaatacac atcacctttt
tagacggcgg 9840 cgctcggttt cttcagcggc caagctggcc ggccaggccg
ccagcttggc atcagacaaa 9900 ccggccagga tttcatgcag ccgcacggtt
gagacgtgcg cgggcggctc gaacacgtac 9960 ccggccgcga tcatctccgc
ctcgatctct tcggtaatga aaaacggttc gtcctggccg 10020 tcctggtgcg
gtttcatgct tgttcctctt ggcgttcatt ctcggcggcc gccagggcgt 10080
cggcctcggt caatgcgtcc tcacggaagg caccgcgccg cctggcctcg gtgggcgtca
10140 cttcctcgct gcgctcaagt gcgcggtaca gggtcgagcg atgcacgcca
agcagtgcag 10200 ccgcctcttt cacggtgcgg ccttcctggt cgatcagctc
gcgggcgtgc gcgatctgtg 10260 ccggggtgag ggtagggcgg gggccaaact
tcacgcctcg ggccttggcg gcctcgcgcc 10320 cgctccgggt gcggtcgatg
attagggaac gctcgaactc ggcaatgccg gcgaacacgg 10380 tcaacaccat
gcggccggcc ggcgtggtgg tgtcggccca cggctctgcc aggctacgca 10440
ggcccgcgcc ggcctcctgg atgcgctcgg caatgtccag taggtcgcgg gtgctgcggg
10500 ccaggcggtc tagcctggtc actgtcacaa cgtcgccagg gcgtaggtgg
tcaagcatcc 10560 tggccagctc cgggcggtcg cgcctggtgc cggtgatctt
ctcggaaaac agcttggtgc 10620 agccggccgc gtgcagttcg gcccgttggt
tggtcaagtc ctggtcgtcg gtgctgacgc 10680 gggcatagcc cagcaggcca
gcggcggcgc tcttgttcat ggcgtaatgt ctccggttct 10740 agtcgcaagt
attctacttt atgcgactaa aacacgcgac aagaaaacgc caggaaaagg 10800
gcagggcggc agcctgtcgc gtaacttagg acttgtgcga catgtcgttt tcagaagacg
10860 gctgcactga acgtcagaag ccgactgcac tatagcagcg gaggggttgg
atcgatccct 10920 gctcgcgcag gctgggtgcc aagctctcgg gtaacatcaa
ggcccgatcc ttggagccct 10980 tgccctcccg cacgatgatc gtgccgtgat
cgaaatccag atccttgacc cgcagttgca 11040 aaccctcact gatccgcatg
cccgttccat acagaagctg ggcgaacaaa cgatgctcgc 11100 cttccagaaa
accgaggatg cgaaccactt catccggggt cagcaccacc ggcaagcgcc 11160
cggacggccg aggtcttccg atctcctgaa gccagggcag atccgtgcac agcacttgcc
11220 gtagaagaac agcaaggccg ccaatgcctg acgatgcgtg gagaccgaaa
ccttgcgctc 11280 gttcgccagc caggacagaa atgcctcgac ttcgctgctg
cccaaggttg ccgggtgacg 11340 cacaccgtgg aaacggatga aggcacgaac
ccagtggaca taagcctgtt cggttcgtaa 11400 gctgtaatgc aagtagcgta
tgcgctcacg caactggtcc agaaccttga ccgaacgcag 11460 cggtggtaac
ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttggggtaca 11520
gtctatgcct cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt
11580 atggagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt
taaacatcat 11640 gagggaagcg gtgatcgccg aagtatcgac tcaactatca
gaggtagttg gcgtcatcga 11700 gcgccatctc gaaccgacgt tgctggccgt
acatttgtac ggctccgcag tggatggcgg 11760 cctgaagcca cacagtgata
ttgatttgct ggttacggtg accgtaaggc ttgatgaaac 11820 aacgcggcga
gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga 11880
gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta
11940 tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc
ttgcaggtat 12000 cttcgagcca gccacgatcg acattgatct ggctatcttg
ctgacaaaag caagagaaca 12060 tagcgttgcc ttggtaggtc cagcggcgga
ggaactcttt gatccggttc ctgaacagga 12120 tctatttgag gcgctaaatg
aaaccttaac gctatggaac tcgccgcccg actgggctgg 12180 cgatgagcga
aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa 12240
aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca
12300 gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc
gcttggcctc 12360 gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa
ggcgagatca ccaaggtagt 12420 cggcaaataa tgtctaacaa ttcgttcaag
ccgacgccgc ttcgcggcgc ggcttaactc 12480 aagcgttaga tgcactaagc
acataattgc tcacagccaa actatcaggt caagtctgct 12540 tttattattt
ttaagcgtgc ataataagcc ctacacaaat tgggagatat atcatgaaag 12600
gctggctttt tcttgttatc gcaatagttg gcgaagtaat cgcaacatcc gcattaaaat
12660 ctagcgaggg ctttactaag ctagcttgct tggtcgttcc ggtaccgtga
acgtcggctc 12720 gattgtacct gcgttcaaat actttgcgat cgtgttgcgc
gcctgcccgg tgcgtcggct 12780 gatctcacgg atcgactgct tctctcgcaa
cgccatccga cggatgatgt ttaaaagtcc 12840 catgtggatc actccgttgc
cccgtcgctc accgtgttgg ggggaaggtg cacatggctc 12900 agttctcaat
ggaaattatc tgcctaaccg gctcagttct gcgtagaaac caacatgcaa 12960
gctccaccgg gtgcaaagcg gcagcgg 12987 25 13226 DNA Artificial
sequence T-DNA vector comprising a chimeric ParG expression
reducing gene 25 cggcaggata tattcaattg taaatggctc catggcgatc
gctctagagg atcttcccga 60 tctagtaaca tagatgacac cgcgcgcgat
aatttatcct agtttgcgcg ctatattttg 120 ttttctatcg cgtattaaat
gtataattgc gggactctaa tcataaaaac ccatctcata 180 aataacgtca
tgcattacat gttaattatt acatgcttaa cgtaattcaa cagaaattat 240
atgataatca tcgcaagacc ggcaacagga ttcaatctta agaaacttta ttgccaaatg
300 tttgaacgat ctgcttcgga tcctagacgc gtgagatcag atctcggtga
cgggcaggac 360 cggacggggc ggtaccggca ggctgaagtc cagctgccag
aaacccacgt catgccagtt 420 cccgtgcttg aagccggccg cccgcagcat
gccgcggggg gcatatccga gcgcctcgtg 480 catgcgcacg ctcgggtcgt
tgggcagccc gatgacagcg accacgctct tgaagccctg 540 tgcctccagg
gacttcagca ggtgggtgta gagcgtggag cccagtcccg tccgctggtg 600
gcggggggag acgtacacgg tcgactcggc cgtccagtcg taggcgttgc gtgccttcca
660 ggggcccgcg taggcgatgc cggcgacctc gccgtccacc
tcggcgacga gccagggata 720 gcgctcccgc agacggacga ggtcgtccgt
ccactcctgc ggttcctgcg gctcggtacg 780 gaagttgacc gtgcttgtct
cgatgtagtg gttgacgatg gtgcagaccg ccggcatgtc 840 cgcctcggtg
gcacggcgga tgtcggccgg gcgtcgttct gggtccatgg ttatagagag 900
agagatagat ttatagagag agactggtga tttcagcgtg tcctctccaa atgaaatgaa
960 cttccttata tagaggaagg gtcttgcgaa ggatagtggg attgtgcgtc
atcccttacg 1020 tcagtggaga tgtcacatca atccacttgc tttgaagacg
tggttggaac gtcttctttt 1080 tccacgatgc tcctcgtggg tgggggtcca
tctttgggac cactgtcggc agaggcatct 1140 tgaatgatag cctttccttt
atcgcaatga tggcatttgt aggagccacc ttccttttct 1200 actgtccttt
cgatgaagtg acagatagct gggcaatgga atccgaggag gtttcccgaa 1260
attatccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt atctttgaca
1320 tttttggagt agaccagagt gtcgtgctcc accatgttga cgaagatttt
cttcttgtca 1380 ttgagtcgta aaagactctg tatgaactgt tcgccagtct
tcacggcgag ttctgttaga 1440 tcctcgattt gaatcttaga ctccatgcat
ggccttagat tcagtaggaa ctaccttttt 1500 agagactcca atctctatta
cttgccttgg tttatgaagc aagccttgaa tcgtccatac 1560 tggaatagta
cttctgatct tgagaaatat gtctttctct gtgttcttga tgcaattagt 1620
cctgaatctt ttgactgcat ctttaacctt cttgggaagg tatttgatct cctggagatt
1680 gttactcggg tagatcgtct tgatgagacc tgctgcgtag gaacgcggcc
gcgtatacgt 1740 atcgatatct tcgaattcga gctcgtcgag cggccgctcg
acgaattaat tccaatccca 1800 caaaaatctg agcttaacag cacagttgct
cctctcagag cagaatcggg tattcaacac 1860 cctcatatca actactacgt
tgtgtataac ggtccacatg ccggtatata cgatgactgg 1920 ggttgtacaa
aggcggcaac aaacggcgtt cccggagttg cacacaagaa atttgccact 1980
attacagagg caagagcagc agctgacgcg tacacaacaa gtcagcaaac agacaggttg
2040 aacttcatcc ccaaaggaga agctcaactc aagcccaaga gctttgctaa
ggccctaaca 2100 agcccaccaa agcaaaaagc ccactggctc acgctaggaa
ccaaaaggcc cagcagtgat 2160 ccagccccaa aagagatctc ctttgccccg
gagattacaa tggacgattt cctctatctt 2220 tacgatctag gaaggaagtt
cgaaggtgaa ggtgacgaca ctatgttcac cactgataat 2280 gagaaggtta
gcctcttcaa tttcagaaag aatgctgacc cacagatggt tagagaggcc 2340
tacgcagcag gtctcatcaa gacgatctac ccgagtaaca atctccagga gatcaaatac
2400 cttcccaaga aggttaaaga tgcagtcaaa agattcagga ctaattgcat
caagaacaca 2460 gagaaagaca tatttctcaa gatcagaagt actattccag
tatggacgat tcaaggcttg 2520 cttcataaac caaggcaagt aatagagatt
ggagtctcta aaaaggtagt tcctactgaa 2580 tctaaggcca tgcatggagt
ctaagattca aatcgaggat ctaacagaac tcgccgtgaa 2640 gactggcgaa
cagttcatac agagtctttt acgactcaat gacaagaaga aaatcttcgt 2700
caacatggtg gagcacgaca ctctggtcta ctccaaaaat gtcaaagata cagtctcaga
2760 agaccaaagg gctattgaga cttttcaaca aaggataatt tcgggaaacc
tcctcggatt 2820 ccattgccca gctatctgtc acttcatcga aaggacagta
gaaaaggaag gtggctccta 2880 caaatgccat cattgcgata aaggaaaggc
tatcattcaa gatctctctg ccgacagtgg 2940 tcccaaagat ggacccccac
ccacgaggag catcgtggaa aaagaagacg ttccaaccac 3000 gtcttcaaag
caagtggatt gatgtgacat ctccactgac gtaagggatg acgcacaatc 3060
ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac
3120 acgctcgagg aatctggctg tctcctaagg gaagtgaaca aggcattttg
tggatttttc 3180 gatcaatcga aacaccatct ctatgcgaag cttttccagg
atttgcacaa caaggatgac 3240 ttttcaagca tcaattccag tgagtacgta
ggagtttcaa caggaaactg gggttgtggt 3300 gcttttggtg gaaaccctga
aatcaagagc atgattcagt ggattgctgc atcacaggct 3360 cttcgccctt
ttgttaatta ctacactttt gagaacgtgt ctctgcaaag attagaggag 3420
gtgatccagt gggtacccca gcttggtaag gaaataatta ttttcttttt tccttttagt
3480 ataaaatagt taagtgatgt taattagtat gattataata atatagttgt
tataattgtg 3540 aaaaaataat ttataaatat attgtttaca taaacaacat
agtaatgtaa aaaaatatga 3600 caagtgatgt gtaagacgaa gaagataaaa
gttgagagta agtatattat ttttaatgaa 3660 tttgatcgaa catgtaagat
gatatactag cattaatatt tgttttaatc ataatagtaa 3720 ttctagctgg
tttgatgaat taaatatcaa tgataaaata ctatagtaaa aataagaata 3780
aataaattaa aataatattt ttttatgatt aatagtttat tatataatta aatatctata
3840 ccattactaa atattttagt ttaaaagtta ataaatattt tgttagaaat
tccaatctgc 3900 ttgtaattta tcaataaaca aaatattaaa taacaagcta
aagtaacaaa taatatcaaa 3960 ctaatagaaa cagtaatcta atgtaacaaa
acataatcta atgctaatat aacaaagcgc 4020 aagatctatc attttatata
gtattatttt caatcaacat tcttattaat ttctaaataa 4080 tacttgtagt
tttattaact tctaaatgga ttgactatta attaaatgaa ttagtcgaac 4140
atgaataaac aaggtaacat gatagatcat gtcattgtgt tatcattgat cttacatttg
4200 gattgattac agttgggaag ctgggttcga aatcgatcac tggatcacct
cctctaatct 4260 ttgcagagac acgttctcaa aagtgtagta attaacaaaa
gggcgaagag cctgtgatgc 4320 agcaatccac tgaatcatgc tcttgatttc
agggtttcca ccaaaagcac cacaacccca 4380 gtttcctgtt gaaactccta
cgtactcact ggaattgatg cttgaaaagt catccttgtt 4440 gtgcaaatcc
tggaaaagct tcgcatagag atggtgtttc gattgatcga aaaatccaca 4500
aaatgccttg ttcacttccc ttaggagaca gccagattct ctagagtcct gctttaatga
4560 gatatgcgag acgcctatga tcgcatgata tttgctttca attctgttgt
gcacgttgta 4620 aaaaacctga gcatgtgtag ctcagatcct taccgccggt
ttcggttcat tctaatgaat 4680 atatcacccg ttactatcgt atttttatga
ataatattct ccgttcaatt tactgattgt 4740 accctactac ttatatgtac
aatattaaaa tgaaaacaat atattgtgct gaataggttt 4800 atagcgacat
ctatgataga gcgccacaat aacaaacaat tgcgttttat tattacaaat 4860
ccaattttaa aaaaagcggc agaaccggtc aaacctaaaa gactgattac ataaatctta
4920 ttcaaatttc aaaaggcccc aggggctagt atctacgaca caccgagcgg
cgaactaata 4980 acgttcactg aagggaactc cggttccccg ccggcgcgca
tgggtgagat tccttgaagt 5040 tgagtattgg ccgtccgctc taccgaaagt
tacgggcacc attcaacccg gtccagcacg 5100 gcggccgggt aaccgacttg
ctgccccgag aattatgcag catttttttg gtgtatgtgg 5160 gccccaaatg
aagtgcaggt caaaccttga cagtgacgac aaatcgttgg gcgggtccag 5220
ggcgaatttt gcgacaacat gtcgaggctc agcaggacct gcaggtcgac ggccgagtac
5280 tggcaggata tataccgttg taatttgtcg cgtgtgaata agtcgctgtg
tatgtttgtt 5340 tgattgtttc tgttggagtg cagcccattt caccggacaa
gtcggctaga ttgatttagc 5400 cctgatgaac tgccgagggg aagccatctt
gagcgcggaa tgggaatgga tttcgttgta 5460 caacgagacg acagaacacc
cacgggaccg agcttcgatc gagcatcaaa tgaaactgca 5520 atttattcat
atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 5580
gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
5640 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
aggttatcaa 5700 gtgagaaatc accatgagtg acgactgaat ccggtgagaa
tggcaaaagt ttatgcattt 5760 ctttccagac ttgttcaaca ggccagccat
tacgctcgtc atcaaaatca ctcgcatcaa 5820 ccaaaccgtt attcattcgt
gattgcgcct gagcgagacg aaatacgccg ctgttaaaag 5880 gacaattaca
aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa 5940
tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttt ccggggatcg
6000 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
gtcggaagag 6060 gcataaattc cgtcagccag tttagtctga ccatctcatc
tgtaacatca ttggcaacgc 6120 tacctttgcc atgtttcaga aacaactctg
gcgcatcggg cttcccatac aatcgataga 6180 ttgtcgcacc tgattgcccg
acattatccg aatctggcaa ttccggttcg cttgctgtcc 6240 ataaaaccgc
ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct 6300
ttgcgcttgc gttttccgga tcttcttgag atcctttttt tctgcgcgta atctgctgct
6360 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
gagctaccaa 6420 ctctttttcc gaaggtaact ggcttcagca gagcgcagat
accaaatact gtccttctag 6480 tgtagccgta gttaggccac cacttcaaga
actctgtagc accgcctaca tacctcgctc 6540 tgctaatcct gttaccagtg
gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 6600 actcaagacg
atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 6660
cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
6720 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
agcggcaggg 6780 tcggaacagg agagcgcacg agggagcttc cagggggaaa
cgcctggtat ctttatagtc 6840 ctgtcgggtt tcgccacctc tgacttgagc
gtcgattttt gtgatgctcg tcaggggggc 6900 ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg gttcctggcc ttttgctggc 6960 cttttgctca
catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 7020
cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
7080 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
tgcggtattt 7140 cacaccgcat atggtgcact ctcagtacaa tctgctctga
tgccgcatag ttaagccagt 7200 atacactccg ctatcgctac gtgactgggt
catggctgcg ccccgacacc cgccaacacc 7260 cgctgacgcg ccctgacggg
cttgtctgct cccggcatcc gcttacagac aagctgtgac 7320 cgtctccggg
agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca 7380
gggtgccttg atgtgggcgc cggcggtcga gtggcgacgg cgcggcttgt ccgcgccctg
7440 gtagattgcc tggccgtagg ccagccattt ttgagcggcc agcggccgcg
ataggccgac 7500 gcgaagcggc ggggcgtagg gagcgcagcg accgaagggt
aggcgctttt tgcagctctt 7560 cggctgtgcg ctggccagac agttatgcac
aggccaggcg ggttttaaga gttttaataa 7620 gttttaaaga gttttaggcg
gaaaaatcgc cttttttctc ttttatatca gtcacttaca 7680 tgtgtgaccg
gttcccaatg tacggctttg ggttcccaat gtacgggttc cggttcccaa 7740
tgtacggctt tgggttccca atgtacgtgc tatccacagg aaagagacct tttcgacctt
7800 tttcccctgc tagggcaatt tgccctagca tctgctccgt acattaggaa
ccggcggatg 7860 cttcgccctc gatcaggttg cggtagcgca tgactaggat
cgggccagcc tgccccgcct 7920 cctccttcaa atcgtactcc ggcaggtcat
ttgacccgat cagcttgcgc acggtgaaac 7980 agaacttctt gaactctccg
gcgctgccac tgcgttcgta gatcgtcttg aacaaccatc 8040 tggcttctgc
cttgcctgcg gcgcggcgtg ccaggcggta gagaaaacgg ccgatgccgg 8100
gatcgatcaa aaagtaatcg gggtgaaccg tcagcacgtc cgggttcttg ccttctgtga
8160 tctcgcggta catccaatca gctagctcga tctcgatgta ctccggccgc
ccggtttcgc 8220 tctttacgat cttgtagcgg ctaatcaagg cttcaccctc
ggataccgtc accaggcggc 8280 cgttcttggc cttcttcgta cgctgcatgg
caacgtgcgt ggtgtttaac cgaatgcagg 8340 tttctaccag gtcgtctttc
tgctttccgc catcggctcg ccggcagaac ttgagtacgt 8400 ccgcaacgtg
tggacggaac acgcggccgg gcttgtctcc cttcccttcc cggtatcggt 8460
tcatggattc ggttagatgg gaaaccgcca tcagtaccag gtcgtaatcc cacacactgg
8520 ccatgccggc cggccctgcg gaaacctcta cgtgcccgtc tggaagctcg
tagcggatca 8580 cctcgccagc tcgtcggtca cgcttcgaca gacggaaaac
ggccacgtcc atgatgctgc 8640 gactatcgcg ggtgcccacg tcatagagca
tcggaacgaa aaaatctggt tgctcgtcgc 8700 ccttgggcgg cttcctaatc
gacggcgcac cggctgccgg cggttgccgg gattctttgc 8760 ggattcgatc
agcggccgct tgccacgatt caccggggcg tgcttctgcc tcgatgcgtt 8820
gccgctgggc ggcctgcgcg gccttcaact tctccaccag gtcatcaccc agcgccgcgc
8880 cgatttgtac cgggccggat ggtttgcgac cgtcacgccg attcctcggg
cttgggggtt 8940 ccagtgccat tgcagggccg gcagacaacc cagccgctta
cgcctggcca accgcccgtt 9000 cctccacaca tggggcattc cacggcgtcg
gtgcctggtt gttcttgatt ttccatgccg 9060 cctcctttag ccgctaaaat
tcatctactc atttattcat ttgctcattt actctggtag 9120 ctgcgcgatg
tattcagata gcagctcggt aatggtcttg ccttggcgta ccgcgtacat 9180
cttcagcttg gtgtgatcct ccgccggcaa ctgaaagttg acccgcttca tggctggcgt
9240 gtctgccagg ctggccaacg ttgcagcctt gctgctgcgt gcgctcggac
ggccggcact 9300 tagcgtgttt gtgcttttgc tcattttctc tttacctcat
taactcaaat gagttttgat 9360 ttaatttcag cggccagcgc ctggacctcg
cgggcagcgt cgccctcggg ttctgattca 9420 agaacggttg tgccggcggc
ggcagtgcct gggtagctca cgcgctgcgt gatacgggac 9480 tcaagaatgg
gcagctcgta cccggccagc gcctcggcaa cctcaccgcc gatgcgcgtg 9540
cctttgatcg cccgcgacac gacaaaggcc gcttgtagcc ttccatccgt gacctcaatg
9600 cgctgcttaa ccagctccac caggtcggcg gtggcccata tgtcgtaagg
gcttggctgc 9660 accggaatca gcacgaagtc ggctgccttg atcgcggaca
cagccaagtc cgccgcctgg 9720 ggcgctccgt cgatcactac gaagtcgcgc
cggccgatgg ccttcacgtc gcggtcaatc 9780 gtcgggcggt cgatgccgac
aacggttagc ggttgatctt cccgcacggc cgcccaatcg 9840 cgggcactgc
cctggggatc ggaatcgact aacagaacat cggccccggc gagttgcagg 9900
gcgcgggcta gatgggttgc gatggtcgtc ttgcctgacc cgcctttctg gttaagtaca
9960 gcgataacct tcatgcgttc cccttgcgta tttgtttatt tactcatcgc
atcatatacg 10020 cagcgaccgc atgacgcaag ctgttttact caaatacaca
tcaccttttt agacggcggc 10080 gctcggtttc ttcagcggcc aagctggccg
gccaggccgc cagcttggca tcagacaaac 10140 cggccaggat ttcatgcagc
cgcacggttg agacgtgcgc gggcggctcg aacacgtacc 10200 cggccgcgat
catctccgcc tcgatctctt cggtaatgaa aaacggttcg tcctggccgt 10260
cctggtgcgg tttcatgctt gttcctcttg gcgttcattc tcggcggccg ccagggcgtc
10320 ggcctcggtc aatgcgtcct cacggaaggc accgcgccgc ctggcctcgg
tgggcgtcac 10380 ttcctcgctg cgctcaagtg cgcggtacag ggtcgagcga
tgcacgccaa gcagtgcagc 10440 cgcctctttc acggtgcggc cttcctggtc
gatcagctcg cgggcgtgcg cgatctgtgc 10500 cggggtgagg gtagggcggg
ggccaaactt cacgcctcgg gccttggcgg cctcgcgccc 10560 gctccgggtg
cggtcgatga ttagggaacg ctcgaactcg gcaatgccgg cgaacacggt 10620
caacaccatg cggccggccg gcgtggtggt gtcggcccac ggctctgcca ggctacgcag
10680 gcccgcgccg gcctcctgga tgcgctcggc aatgtccagt aggtcgcggg
tgctgcgggc 10740 caggcggtct agcctggtca ctgtcacaac gtcgccaggg
cgtaggtggt caagcatcct 10800 ggccagctcc gggcggtcgc gcctggtgcc
ggtgatcttc tcggaaaaca gcttggtgca 10860 gccggccgcg tgcagttcgg
cccgttggtt ggtcaagtcc tggtcgtcgg tgctgacgcg 10920 ggcatagccc
agcaggccag cggcggcgct cttgttcatg gcgtaatgtc tccggttcta 10980
gtcgcaagta ttctacttta tgcgactaaa acacgcgaca agaaaacgcc aggaaaaggg
11040 cagggcggca gcctgtcgcg taacttagga cttgtgcgac atgtcgtttt
cagaagacgg 11100 ctgcactgaa cgtcagaagc cgactgcact atagcagcgg
aggggttgga tcgatccctg 11160 ctcgcgcagg ctgggtgcca agctctcggg
taacatcaag gcccgatcct tggagccctt 11220 gccctcccgc acgatgatcg
tgccgtgatc gaaatccaga tccttgaccc gcagttgcaa 11280 accctcactg
atccgcatgc ccgttccata cagaagctgg gcgaacaaac gatgctcgcc 11340
ttccagaaaa ccgaggatgc gaaccacttc atccggggtc agcaccaccg gcaagcgccc
11400 ggacggccga ggtcttccga tctcctgaag ccagggcaga tccgtgcaca
gcacttgccg 11460 tagaagaaca gcaaggccgc caatgcctga cgatgcgtgg
agaccgaaac cttgcgctcg 11520 ttcgccagcc aggacagaaa tgcctcgact
tcgctgctgc ccaaggttgc cgggtgacgc 11580 acaccgtgga aacggatgaa
ggcacgaacc cagtggacat aagcctgttc ggttcgtaag 11640 ctgtaatgca
agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc 11700
ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag
11760 tctatgcctc gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg
tttgatgtta 11820 tggagcagca acgatgttac gcagcagggc agtcgcccta
aaacaaagtt aaacatcatg 11880 agggaagcgg tgatcgccga agtatcgact
caactatcag aggtagttgg cgtcatcgag 11940 cgccatctcg aaccgacgtt
gctggccgta catttgtacg gctccgcagt ggatggcggc 12000 ctgaagccac
acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca 12060
acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag
12120 attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc
gtggcgttat 12180 ccagctaagc gcgaactgca atttggagaa tggcagcgca
atgacattct tgcaggtatc 12240 ttcgagccag ccacgatcga cattgatctg
gctatcttgc tgacaaaagc aagagaacat 12300 agcgttgcct tggtaggtcc
agcggcggag gaactctttg atccggttcc tgaacaggat 12360 ctatttgagg
cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc 12420
gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa
12480 atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc
ccagtatcag 12540 cccgtcatac ttgaagctag acaggcttat cttggacaag
aagaagatcg cttggcctcg 12600 cgcgcagatc agttggaaga atttgtccac
tacgtgaaag gcgagatcac caaggtagtc 12660 ggcaaataat gtctaacaat
tcgttcaagc cgacgccgct tcgcggcgcg gcttaactca 12720 agcgttagat
gcactaagca cataattgct cacagccaaa ctatcaggtc aagtctgctt 12780
ttattatttt taagcgtgca taataagccc tacacaaatt gggagatata tcatgaaagg
12840 ctggcttttt cttgttatcg caatagttgg cgaagtaatc gcaacatccg
cattaaaatc 12900 tagcgagggc tttactaagc tagcttgctt ggtcgttccg
gtaccgtgaa cgtcggctcg 12960 attgtacctg cgttcaaata ctttgcgatc
gtgttgcgcg cctgcccggt gcgtcggctg 13020 atctcacgga tcgactgctt
ctctcgcaac gccatccgac ggatgatgtt taaaagtccc 13080 atgtggatca
ctccgttgcc ccgtcgctca ccgtgttggg gggaaggtgc acatggctca 13140
gttctcaatg gaaattatct gcctaaccgg ctcagttctg cgtagaaacc aacatgcaag
13200 ctccaccggg tgcaaagcgg cagcgg 13226
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